Compositions and methods for membrane protein detergent stability screen

ABSTRACT

The invention provides methods to assess protein stability and to obtain sizing information. In one aspect, the screen comprises a 94 detergent panel and a series of MWCO filtered microplates. A protein of interest is bound to an affinity matrix and aliquoted into a 96-well microplate. Wells containing the immobilized protein are washed in the new detergent and then eluted in the new detergent into a collection plate. Protein not stable in the new detergent is precipitated on the resin and not present in the elutions. Half of the elution is passed through a high (i.e., 300 kDa) MWCO microplate and the other half through a low (i.e., 100 kDa) MWCO microplate. Elutions from the microplates are spotted on a nitrocellulose membrane, visualized by Western analysis (or by some other method), and quantified. The high MWCO provides stability readout and the ratio of low/high kDa provides sizing information.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/221,198 filed Jun. 29, 2009, thedisclosure of which is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with United States Government supportunder Grant No. 5R01 GM075931, awarded by The National Institutes ofHealth. The United States Government has certain rights in theinvention.

BACKGROUND

Membrane proteins comprise between 15% and 39% of the human proteome and45% of drugs target these proteins. Membrane proteins are prevalent inthe proteomes of pathogenic microorganisms and are the targets of manyantimicrobial agents. Membrane proteins play essential roles inpathophysiology and the biology of all organisms. Near atomic resolutionstructures are required for our understanding of the function of thesemolecules. X-ray crystallography, electron crystallography, and nuclearmagnetic resonance spectroscopy (NMR) are the currently availablemethods for obtaining high resolution structures of macromolecules,including membrane proteins.

Purified membrane proteins require surfactants, typically detergents, toremain soluble in an aqueous environment. The complex of the membraneprotein and the associated detergent molecules (the protein detergentcomplex, PDC), is the object studied by x-ray crystallography or NMR.

The difficulties of working with membrane proteins are demonstrated bythe fact that membrane protein structures represent less than 1% of thetotal number of protein structures in the Protein Data Bank, despiteintegral membrane proteins encompassing 15-30% of most genomes [1; 2;3]. Technical challenges in membrane protein structure determinationinclude expression (to obtain suitable amounts of protein), purification(to obtain suitably stable and functional protein), and samplepreparation (to obtain suitable two-dimensional crystals for electroncrystallography, three-dimensional crystals for x-ray crystallography,or solutions for NMR spectroscopy).

In preparation for structural (and other) studies, membrane proteins areextracted from their native lipid bilayer environment, and this membranebilayer is replaced by a membrane-mimetic. The membrane-mimetic soluteis almost always a detergent at a concentration above its criticalmicelle concentration (CMC), where the detergent surrounds thehydrophobic membrane-facing portion of the membrane protein and formsthe protein-detergent complex (PDC). PDCs are in equilibrium withdetergent micelles and monomers in this solution. The chemical-space ofdetergents is large, and the solution (and crystallization) propertiesof a membrane protein are intimately related to the properties of thedetergent(s) comprising the PDC [4; 5]. In addition, the function of amembrane protein can be maintained at native or near-native levels orcan be completely abrogated, depending upon the detergent composition ofthe PDC.

Currently, according to the Membrane Proteins of Known Structuredatabase, 231 unique integral membrane protein structures have beensolved by x-ray crystallography. The Membrane Protein Data Bank database[6] lists 864 non-unique membrane protein x-ray crystal structures, forwhich more than fifty different detergents have been used in theirsolubilization and/or crystallization. These detergents are not equallyrepresented. For example, five detergents, n-dodecyl-β-D-maltopyranoside(DDM); n-decyl-β-D-maltopyranoside (DM); n-nonyl-β-D-glucopyranoside(NG); n-octyl-β-D-glucopyranoside (OG); andn-dodecyl-N,N-dimethylamine-N-oxide (LDAO) have yielded the majority ofα-helical membrane protein structures [7].

While this speaks to the utility (and extensive use) of these fivedetergents, over 40% of the structures solved to date requireddetergents other than those five. As such, survey of membrane proteinstability in “detergent-space” is an important aspect of membraneprotein structural biology (and biochemistry).

There are several methods to test detergent solubility of membraneproteins. These methods include: gel filtration; dilution [5]; and theultracentrifugation dispersity sedimentation assay [8]. Inspection ofthe gel filtration chromatogram has been routinely used for both solubleand membrane proteins to assess the quality of a protein.

The method of fluorescence-detection size-exclusion chromatography(FSEC) was an advance in gel filtration chromatography of integralmembrane proteins [9]. The unique optical signal of afluorescently-tagged recombinant protein enables that protein to bedetected and characterized in a solubilized mixture, prior topurification. Also, the use of fluorescence (versus absorbance)detection increases the sensitivity by several orders of magnitude,requiring less solubilized (or purified) protein for the chromatographyanalysis. In order to evaluate detergent stability, gel filtration canbe performed in either of two ways: 1) the column is equilibrated in thedetergent to be tested and the protein is loaded onto the column(“detergent-specific mobile phase”) or 2) the protein is exchanged intoa new detergent and then injected onto a column equilibrated with aknown “good” detergent for all chromatographic runs (“generic mobilephase”).

The use of the generic mobile phase speeds up the gel filtration runs byeliminating the column washing and equilibration steps for the nextdetergent. The generic mobile phase method rests upon the assumptionthat if a protein sample has been exchanged into an incompatibledetergent, then a compatible detergent in the mobile phase will notreverse the deleterious effects of that incompatible detergent [9].

Data from our lab suggests that this is not true for all cases, so we donot currently favor the generic mobile phase method. We note that theoriginal FSEC publication [9] utilizes a generic mobile phase; however,fluorescence detection is equally applicable to use of adetergent-specific mobile phase. For the dilution method, concentratedprotein is diluted into a new test detergent and the Abs₃₂₀ nm:Abs₂₈₀ nmratio is recorded over time. Because Abs₃₂₀ nm is indicative of proteinaggregation, an increase in this ratio is diagnostic of the protein notbeing stable in the new detergent [5]. In the ultracentrifugationdispersity sedimentation assay, the protein is concentrated, dilutedinto the test detergent buffer with three concentration/dilution steps,and finally allowed to incubate overnight. At that point, a sample istaken while the rest of the protein is spun in the ultracentrifuge topellet any aggregated protein. Another protein sample is taken afterultracentrifugation and both the pre- and post-ultracentrifugationsamples are run on SDS-PAGE and compared. Any difference in bandintensity between the two samples is indicative of aggregated proteinbeing removed during the intermediate ultracentrifugation step and thusrelated to detergent stability [8].

These three methods all possess shortcomings. The biggest limitation isthat the methods described above are not detergent exchanges, but ratherare detergent dilutions (the exception is the single case where theprotein is already in the same detergent as that present in the gelfiltration mobile phase). This is a problem if the initial detergent isnot diluted to a concentration below its CMC or, in the case of gelfiltration, if the original detergent's micelles are not separated fromthe protein-detergent complex (PDC), or if a mixed detergent populationexists. In these instances, the presence of the original detergent can“protect” a protein from a destabilizing detergent resulting in falsepositives.

The original detergent's concentration is of great concern especiallywhen the method utilizes an ultrafiltration concentration step of theprotein since detergent micelles typically concentrate along with theprotein even when a large molecular weight cut-off (MWCO) is used.Another limitation is that milligram amounts of protein and largeamounts of expensive detergent reagents may be necessary, especially ifthere are a large number of conditions to be tested. Lastly, the timerequired to perform each method can be long, which usually limits thenumber of detergents surveyed, especially in the case of gel filtrationwhere only one detergent can be tested at a time.

There is a long felt need in the art for compositions and methods usefulas a system for efficiently determining conditions and the properdetergents for membrane proteins from solutions containing a membraneprotein in a purified and soluble state. The present invention satisfiesthese needs.

SUMMARY OF THE INVENTION

The present invention pertains to the field of membrane proteinbiochemistry and structural biology. The assay technology developed andpresented here focuses upon the selection of appropriate detergents foruse with a specific membrane protein, which is a critical aspect of bothpurification and sample preparation. The screen of the present inventionwas developed to overcome shortcomings of current methods for detergentscreening as well to expand the number of detergents examined.

The present invention encompasses a quick and robust method to assessmembrane protein stability and to obtain rudimentary sizing informationin a high throughput format. Various aspects and embodiments of theinvention are described in further detail below.

The present invention provides compositions and methods useful for a newmembrane protein detergent screening assay to determine stability andsize of the protein.

The present invention provides a method for determining the stabilityand size of a protein in a test detergent. In one aspect, the methodcomprises obtaining a solution comprising a protein of interest in afirst detergent and then adding an effective amount of an affinity resinto the solution. Then an aliquot of the solution comprising the proteinin the first detergent and the affinity resin is added to a firstchamber or well, wherein the chamber or well has a filter in it with apore size of about 0.2 μm. Then, as a means of detergent exchange a washsolution comprising a different detergent (i.e., a test detergent) isused to wash away the first detergent in the first chamber. Up to about20 column volumes of the different test detergent wash solution can beused. Then, the protein is eluted through the filter by using an elutionsolution prepared using the different test detergent and eluting theprotein with up to about 6 column volumes of the elution solution. Analiquot of the eluate is then passed through a second chamber or wellcomprising a high molecular weight cut-off filter and another aliquot ofthe eluate is passed through a third chamber comprising a low molecularweight cut-off filter. Then, the amount of protein in each of the twoeluates passing through the high and low molecular weight cut-offfilters is determined and then compared by comparing the amount ofprotein eluted through the high molecular weight cut-off filter with theamount of protein eluted through the low molecular weight cut-offfilter, thereby determining the stability and size of a protein in atleast one detergent.

In one aspect, the filtration of the detergent mixtures in the chambersor wells can be enhanced by centrifuging the chambers or wells.

For determining sizing information of a protein, the present inventionprovides a differential filtration (DF) process using filters withdifferent molecular weight cut-offs. In one embodiment, the highmolecular weight cut-off filter is about 300 kDa and the low molecularweight cut-off filter is about 300 kDa.

In one embodiment, the chamber or well is part of a multiwell plate.

In one embodiment, the present invention provides a multiwellplate-based detergent screening assay, which is coupled with an assayfor determining molecular weight ranges of the protein of interest. Inone aspect, the multiwell plate is a microplate. The practice of theinvention is not limited to a specific number of wells per plate. Aplate may comprise multiple wells or chambers comprising an appropriatefilter(s) for the process being performed. For example, the plate can bea 1 well, 6 well, 12 well, 24 well, 48 well, 96 well, 384 well, or 1536well plate. In one aspect, the 96 well plate is an SBS format plate.

Multiple detergents can be used in the screening process and the numberof detergents tested can be modified based on the number of wells orchambers in the plate or container being used (See Table 1).

In one embodiment, the assay comprises a panel of 94 detergents suitablefor structural studies on membrane proteins and a set of labware thatallows for the determination of both stability and rudimentary size theprotein:detergent complex (PDC) to be obtained. This is a highthroughput assay that utilizes microgram amounts and microliter volumesof reagents to obtain detergent stability information in approximately 2hours. The present invention also encompasses using a different numberof detergents than 94.

The method further allows for the use of low levels of protein. In oneembodiment, about 1000 micrograms or less of a protein of interest isneeded in the first detergent. In one aspect, about 500 micrograms orless, or about 400 micrograms or less, or about 200 micrograms or less,or about 100 micrograms or less, or about 50 micrograms or less of theprotein can be used. Furthermore, if an appropriate, protein-specificdetection method is used, the protein of interest can be screened in acrude, unpurified form.

The method is rapid and in some cases can be performed in less thanabout two hours. Protein amounts eluted through the high and lowmolecular weight can be determined by, for example, dot blot or Westernblot analysis, wherein the amounts are measured and quantified byestablished techniques and methods. In one aspect, the high molecularweight cut-off dot blots, are normalized and plotted with the ratio oflow:high normalized intensities and the values grouped into quartiles.In another aspect, the protein amounts determined from the highmolecular weight cut-off dot blots are normalized and plottedgraphically on the abscissa while the ratio of low:high normalizedintensities are plotted on the ordinate.

The DF method alone can provide rudimentary sizing information ofmacromolecules without the need for gel filtration. DF can be performedusing the described microplates, different sets of MWCO plates, or otherformats in which MWCO filters are utilized (e.g., spin columns,ultrafiltration cell).

The compositions and methods of the invention are also useful, forexample, to screen detergent mixtures, additives, ionic strength, and pHfor soluble proteins as well.

In one aspect, the compositions and methods of the invention are usefulfor membrane proteins.

In one embodiment, the concentrations of the different detergents usedare based on the critical micelle concentrations of the differentdetergents. In one aspect, the concentration used for a detergent is thecritical micelle concentration.

In one embodiment, a detergent used in the invention has moderate orhigh aqueous solubility. In one embodiment, a detergent used in theinvention has zwitterionic or nonionic headgroups.

The present invention further encompasses the preparation and use of adetergent panel and a kit using the detergents and controls describedherein. In one aspect, other detergents can also be used. Additional kitand panel details are as follows:

Detergent Screening Kit—all of the necessary reagents to perform thedetergent stability assay

-   -   a. Detergent Stability Panel—microplate block containing the        reagents of the detergent panel in 2× working concentrations.    -   b. DF Microplates—set of filtered microplates required to        perform the assay. The low and high plates display retention and        passage properties suited to the practice of the present        invention, but other plates and other molecular weight exclusion        limits are encompassed as well. The present invention is not        limited to the used of these two molecular weight cutoffs and        that is why the terms “high” and “low” are also used, to ensure        that the emphasis is on the fact that the plates are of        different molecular weight cut-offs.

In one embodiment, at least one of the following detergents is used in akit or panel or in the methods of the invention:

ZWITTERGENT® 3-12, ZWITTERGENT® 3-14, n-Decyl-N,N-dimethylglycine,n-Dodecyl-N,N-dimethylglycine, n-Decyl-N,N-dimethylamine-N-oxide,n-Undecyl-N,N,-dimethylamine-N-oxide,n-Dodecyl-N,N-dimethylamine-N-oxide, C-DODECAFOS™, CYCLOFOS™-4,CYCLOFOS™-5, CYCLOFOS™-6, CYCLOFOS™-7, FOS-CHOLINE®-10, FOS-CHOLINE®-11,FOS-CHOLINE®-12, FOS-CHOLINE®-13, FOS-CHOLINE®-14, FOS-CHOLINE®-ISO-11,FOS-CHOLINE®-ISO-11-6U, FOS-CHOLINE®-ISO-9, FOS-CHOLINE®-UNSAT-11-10,1,2-Diheptanoyl-sn-glycero-3-phosphocholine, LysoPC-10, LysoPC-12,FOSFEN™-9, CHAPS, CHAPSO, n-Dodecyl-N,N-(dimethylammonio)undecanoate,n-Dodecyl-N,N-(dimethylammonio)butyrate, LAPAO, TRIPAO, TWEEN® 20,BRIJ®35, TRITON® X-100, TRITON® X-114, TRITON® X-305, TRITON® X-405,[Octylphenoxy]polyethoxyethanol, Dimethyloctylphosphine oxide,Dimethylnonylphosphine oxide, Dimethyldecylphosphine oxide,Dimethylundecylphosphine oxide, Dimethyldodecylphosphine oxide,Triethylene glycol monohexyl ether, Tetraethylene glycol monohexylether, Pentaethylene glycol monohexyl ether, Pentaethylene glycolmonoheptyl ether, Tetraethylene glycol monooctyl ether, Pentaethyleneglycol monooctyl ether, Hexaethylene glycol monooctyl ether,Pentaethylene glycol monodecyl ether, Hexaethylene glycol monodecylether, Polyoxyethylene(9)decyl ether, Octaethylene glycol monododecylether, Polyoxyethylene(9)dodecyl ether, Polyoxyethylene(10)dodecylether, Polyoxyethylene(8)tridecyl ether, Big CHAP, Big CHAP, deoxy,Genapol® X-100, n-Heptyl-β-D-thioglucopyranoside,n-Octyl-β-D-glucopyranoside, n-Nonyl-β-D-glucopyranoside, CYGLU®-3,HECAMEG, Hega®-9, C-Hega®-10, C-Hega®-11, CYMAL®-3, CYMAL®-4, CYMAL®-5,CYMAL®-6, CYMAL®-7, 2,6-Dimethyl-4-heptyl-β-D-maltoside,n-Octyl-β-D-maltopyranoside, n-Nonyl-β-D-maltopyranoside,n-Decyl-α-D-maltopyranoside, n-Decyl-β-D-maltopyranoside,n-Undecyl-α-D-maltopyranoside, n-Undecyl-β-D-maltopyranoside,ω-Undecylenyl-β-D-maltopyranoside, n-Dodecyl-α-D-maltopyranoside,n-Dodecyl-β-D-maltopyranoside, n-Tridecyl-β-D-maltopyranoside,n-Octyl-β-D-thiomaltopyranoside, n-Nonyl-β-D-thiomaltopyranoside,n-Decyl-β-D-thiomaltopyranoside, n-Undecyl-β-D-thiomaltopyranoside,n-Dodecyl-β-D-thiomaltopyranoside, Sucrose8, Sucrose10, and Sucrose12.

In one embodiment, a detergent panel for determining the stability andsize of a protein is provided. In one aspect, the panel comprises atleast two detergents at or above their critical micelle concentrations,and optionally a positive control and a negative control. In one aspect,the detergents are selected from Table 1. In one aspect, the detergentsand controls of Table 1 are used. In one aspect, the stockconcentrations provided vary with the CMC value of the detergent, andmay for example be 2×, 2.5×, 3×, 10×, 50×, and 100×, as disclosed in theExamples.

In one embodiment, all of the test detergents described above are testedor provided in a kit or panel. In another embodiment, other detergentsnot disclosed herein can be used in the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Flowchart of the detergent stability assay developed hereincalled Differential Filtration Assay (“DFA”), previously referred to asPrompt Assay of Stability and Size (“PASS”). A generic protocol forperforming the assay is presented. Note that the specific compositionsfor the wash solution of the third step (“Wash each well with 20CV newdetergent”) and for the elution solution the fourth step (“Elute proteinin 6CV new detergent) are described in the Materials and Methods sectionunder “Detergent stability assay”.

FIG. 2—Graphically depicts filter plate flow through of molecular weightstandards. The Abs₂₈₀ nm was measured for each stock solution, and theeluate from each plate in triplicate. The left set of bars represent 0.2μm GHP, the center set of bars represents the High MWCO (molecularweight cut-off) group, and the right set of bars represents the Low MWCOgroup. Bars represent Blue Dextran, Thryoglobin, Apoferritin, Catalyase,b-Amylase, Aldolase, Alcohol Dehydrogenase, Albumin, Carbonic Anhydrase,and Cytochrome C. The Ordinate represent Percent Flow Through. Thepercent difference between the eluate and the stock is presented on thegraph. The error bars represent the error propagated from the standarddeviation of each triplicate measurement. No passage of blue dextran wasobserved in either the high or low MWCO filter plates.

FIG. 3 (FIGS. 3A-3D)—Dot blots of eluted protein after exchange into thenew detergent. 10 μl of the elutions from the high and low MWCO plateswere spotted on nitrocellulose membrane and visualized by Western blotusing an IRDye®800CW conjugated His-tag antibody and a LI-COR Odysseyimaging system. The dot intensities were quantified with medianbackground correction within the Odyssey software. The two left panelsrepresent High MWCO plates (FIGS. 3A and 3C) and the two right panelsrepresent Low MWCO plates (FIGS. 3B and 3D). The two upper plates (FIGS.3A and 3B) represent AqpZ and the two lower plates (FIGS. 3C and 3D)represent KcsA.

FIG. 4—Stability and relative size bar graphs. The normalizedintensities from the high MWCO dot blots are plotted along with theratio of low:high (low/high) normalized intensities for AqpZ (FIG. 4A)and KcsA (FIG. 4B). The values are grouped into quartiles, indicated bythe gridlines. The high intensity is directly proportional to stabilitywhile low/high is inversely proportional to the particle size. Non-realratio values (i.e. low intensity greater than high intensity) are givenin parenthesizes. These non-real ratio values are all from high and lowintensities within the same quartile rank except those indicated by anasterisk (*).

FIG. 5—Quartile grid plot. The normalized intensities from the high MWCOdot blots are plotted on the horizontal axis while the ratios oflow:high (low/high) normalized intensities for AqpZ (FIG. 5A) and KcsA(FIG. 5B) are plotted on the vertical axis. The well numbers are shownnext to each dot. Non-real ratio values (i.e. low intensity greater thanhigh intensity) are located in the grayed out area of the plot. Thesenon-real ratio values are all from high and low intensities within thesame quartile rank except those indicated by an asterisk (*).

FIG. 6—Low MWCO elution intensity correlates to HPSEC retention time.Larger amounts of AqpZ (FIG. 6A) and KcsA (FIG. 6B) were detergentexchanged using spin columns and then 10 μl injected onto a calibratedSuperdex™ 200 5/150 GL gel filtration column equilibrated in theexchanged buffer. The retention times for MW gel filtration standardsare shown on each chromatogram. The insets show the dot blot spots foreach detergent. The ordinate represents normalized fluorescence and theabscissa represents retention time (in minutes).

DETAILED DESCRIPTION Abbreviations and Acronyms

-   -   AqpZ—Aquaporin Z    -   B₂₂—second virial coefficient    -   CMC—critical micelle concentration    -   CV—column volume    -   DF—differential filtration    -   DFA—differential filtration assay (also see PASS)    -   DM—n-β-D-maltopyranoside    -   DTT—dithiothreitol    -   FID—free interface diffusion    -   F SEC—fluorescence-detection size-exclusion chromatography    -   GHP—GH polypro hydrophobic polypropylene    -   IMAC—immobilized metal affinity chromatography    -   LDAO—n-dodecyl-N,N-dimethylamine-N-oxide    -   min—minute(s)    -   MW—molecular weight    -   MWCO—molecular weight cutoff    -   OG—n-octyl-β-D-glucoside    -   NMR—nuclear magnetic resonance spectroscopy    -   Membrane Protein Detergent Stability Screen, also referred to as        the Prompt Assay of Stability and Size (PASS)—PASS    -   PASS—prompt assay of stability and size (also see DFA)    -   MPEG—polyethylene glycol monomethylether    -   PDC—protein detergent complex    -   PEG—polyethylene glycol    -   PES—polyethersulfone    -   SBS—Society for Biomolecular Sciences    -   SEC—Size Exclusion Chromatography    -   SEC-M—Size Exclusion Chromatography-Mimetic    -   TCA—trichloroacetic acid    -   TCEP—tris(2-carboxyethyl) phosphine hydrochloride

DEFINITIONS

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below. Unlessdefined otherwise, all technical and scientific terms used herein havethe commonly understood meaning by one of ordinary skill in the art towhich the invention pertains. Although any methods and materials similaror equivalent to those described herein may be useful in the practice ortesting of the present invention, preferred methods and materials aredescribed below. Specific terminology of particular importance to thedescription of the present invention is defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. For example, in oneaspect, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20%.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The expression “amino acid” as used herein is meant to include bothnatural and synthetic amino acids, and both D and L amino acids.“Standard amino acid” means any of the twenty standard L-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acidresidue” means any amino acid, other than the standard amino acids,regardless of whether it is prepared synthetically or derived from anatural source. As used herein, “synthetic amino acid” also encompasseschemically modified amino acids, including but not limited to salts,amino acid derivatives (such as amides), and substitutions. Amino acidscontained within the peptides of the present invention, and particularlyat the carboxy- or amino-terminus, can be modified by methylation,amidation, acetylation or substitution with other chemical groups whichcan change the peptide's circulating half-life without adverselyaffecting their activity. Additionally, a disulfide linkage may bepresent or absent in the peptides of the invention.

The term “amino acid” is used interchangeably with “amino acid residue,”and may refer to a free amino acid and to an amino acid residue of apeptide. It will be apparent from the context in which the term is usedwhether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

The nomenclature used to describe the peptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino- and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.

The term “basic” or “positively charged” amino acid, as used herein,refers to amino acids in which the R groups have a net positive chargeat pH 7.0, and include, but are not limited to, the standard amino acidslysine, arginine, and histidine.

As used herein, an “analog” of a chemical compound is a compound that,by way of example, resembles another in structure but is not necessarilyan isomer (e.g., 5-fluorouracil is an analog of thymine).

A “compound,” as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above. When referring to acompound of the invention, and unless otherwise specified, the term“compound” is intended to encompass not only the specified molecularentity but also its pharmaceutically acceptable, pharmacologicallyactive analogs, including, but not limited to, salts, polymorphs,esters, amides, prodrugs, adducts, conjugates, active metabolites, andthe like, where such modifications to the molecular entity areappropriate.

A “chamber”, as used herein, refers to something to which a solution canbe added, such as a tube or well of a multiwell plate, etc.

By the phrase “contacting a sample of a protein with at least oneformulation” means that if more than one formulation is tested that thesample of the protein is either tested as an aliquot from the sample orthat separate samples of the protein are prepared and used.

The use of the word “detect” and its grammatical variants is meant torefer to measurement of the species without quantification. The terms“detect” and “identify” are used interchangeably herein.

An “effective amount of an affinity resin”, as used herein, is an amountof resin needed to bind a protein in the detergent.

A “filter plate” as used herein, refers to a plate or other such device,where the bottom or an aspect of the well(s) or chamber(s) comprises afilter. For example, the plate can be a multiwell plate where the bottomof each well is a filter. The practice of the invention is not limitedto the use of just those filter plates described herein.

As used in the specification and the appended claims, the terms “forexample,” “for instance,” “such as,” “including” and the like are meantto introduce examples that further clarify more general subject matter.Unless otherwise specified, these examples are provided only as an aidfor understanding the invention, and are not meant to be limiting in anyfashion.

A “formulation sample” or “sample of a formulation” means an aliquot ofthe designated formulation. When referenced in a kit, the aliquot isenough to perform at least one experiment for a protein sample. In oneaspect, the formulation sample may be in a quantity such that multipleexperiments can be performed. A kit may also contain multiple samples ofeach formulation.

As used herein, the term “high” or “high plate” refers to a molecularweight cut-off plate which has a higher molecular weight cut-off thanthe companion low molecular weight cut-off plate with which it is pairedfor the size determination steps of the invention.

As used herein, the term “low” or “low plate” refers to a molecularweight cut-off plate which has a lower molecular weight cut-off than thecompanion high molecular weight cut-off plate with which it is pairedfor the size determination steps of the invention.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the formulations and methods ofthe invention in the kit. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains theidentified formulations of invention or be shipped together with acontainer which contains the identified compound. Alternatively, theinstructional material may be shipped separately from the container withthe intention that the instructional material and the formulations beused cooperatively by the recipient.

By the term “optionally centrifuging” a filter plate, well, or chamberis meant centrifuging it to enhance filtration.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

The terms “solid support”, “surface” and “substrate” are usedinterchangeably and refer to a structural unit of any size, where saidstructural unit or substrate has a surface suitable for immobilizationof molecular structure or modification of said structure and saidsubstrate is made of a material such as, but not limited to, metal,metal films, glass, fused silica, synthetic polymers, and membranes.

The term “standard,” as used herein, refers to something used forcomparison. For example, it can be a known standard agent or compoundwhich is administered and used for comparing results when administeringa test compound, or it can be a standard parameter or function which ismeasured to obtain a control value when measuring an effect of an agentor compound on a parameter or function. “Standard” can also refer to an“internal standard”, such as an agent or compound which is added atknown amounts to a sample and which is useful in determining such thingsas purification or recovery rates when a sample is processed orsubjected to purification or extraction procedures before a marker ofinterest is measured.

EMBODIMENTS

Structural studies on integral membrane proteins are routinely performedon protein-detergent complexes (PDCs) consisting of purified proteinsolubilized in a particular detergent. Of all of the membrane proteinscrystal structures solved to date, a subset of only four detergents hasbeen used in over half of these structures.

Unfortunately, many membrane proteins are not well-behaved in these fourdetergents and/or fail to yield well-diffracting crystals.Identification of detergents that maintain the solubility and stabilityof a membrane protein is a critical step, and can be a lengthy and“protein-expensive” process. There is an unmet need for compositions andmethods useful for screening multiple detergents and determining thosebest suitable for proteins to obtain well-diffracting crystals. Thepresent application discloses an assay that characterizes the stabilityand size of membrane proteins exchanged into a panel of 94 commerciallyavailable and chemically diverse detergents. DFA, utilizing a set of SBS-format filtered microplates, requires sub-milligram quantities ofpurified protein, small quantities of detergents and other reagents, andis performed in its entirety in several hours.

When membrane proteins are removed from their lipid-rich, hydrophobicenvironment into an aqueous environment, surfactants/detergents aretypically used to maintain their structure and activity by mimickingtheir native environment.

The current methods employed by others in the field include:

1) Traditional Gel Filtration Chromatography—inject a purified membraneprotein onto a column equilibrated into a test detergent and evaluatethe “goodness” of the elution chromatogram. This method is timeconsuming, material expensive, and is not a detergent exchange but adilution method.

2) Generic Mobile Phase Gel Filtration Chromatography—this is the sameas the previous method except the protein is first diluted into the testdetergent and then injected onto a column equilibrated in a known “good”detergent. The premise is that if the new detergent destabilizes theprotein, the good detergent will not rescue it and produce a goodchromatogram. We have found in our lab that this is not true for allcases so we do not subscribe to this method. This method is quicker thantraditional gel filtration in that there is no column equilibration stepbut it still suffers from the other shortcomings.

3) Abs₃₂₀ nm/Abs₂₈₀ nm Ratio—the protein is concentrated and thendiluted 10-100 fold into a new detergent. An increase in the Abs₃₂₀value is indicative of precipitated protein and thus an incompatibledetergent. This method has a high throughput capability but theconcentration step required to give a meaningful Abs₂₈₀ value afterdilution allows for the original detergent to be concentrated as wellduring the concentration step. This could prevent the original detergentto not be diluted below its CMC value and thus protect the protein fromthe new detergent, resulting in a false positive.

4) Ultracentrifugation/SDS PAGE—the protein is diluted and concentratedin a new detergent several times using a centripetal concentrator. Analiquot of this sample is taken for SDS-PAGE while the rest is spun inan ultracentrifuge to pellet any precipitated protein. Another SDS-PAGEsample is made from the supernate of the ultracentrifugation run andboth are run on an SDS-PAGE protein gel. Differences in band intensitybefore and after the ultracentrifugation step are indicative of anincompatible detergent. This method suffers from the same shortcoming asthe previous method due to the centripetal concentration step. Also, theultracentrifugation step is not amenable to high throughput.

However, the method of the present invention overcomes the speedlimitations of other methods by simultaneously examining multiplesamples, in one aspect 94 samples, in parallel using high throughputmethods, small amounts of materials (both protein and detergents) arerequired, and this method is an exchange method so there is no worryabout the original detergent not being diluted below its CMC value.

In order to obtain sizing information on the protein:detergent complex(PDC) without using gel filtration chromatography we implemented the useof the MWCO filtered microplates to create a Size ExclusionChromatography Mimetic, or SEC-M, also referred to as DF. Utilizing twodifferent molecular weight cut-offs, such as 300 kDa and 100 kDa plates,one can obtain rudimentary sizing information of the PDC in a fractionof the time it would take to run all the samples over gel filtration.

The present invention provides compositions and methods useful forquickly determining the stability and size of a protein by usingmultiple detergents and methods for applying detergent solutionscomprising a protein to a series filters with varied molecular weightcut-offs, and allows for the selection of an appropriate detergent foruse with the protein. In one aspect, the protein is a membrane protein.

In one embodiment, the present invention provides a first step, where abuffer exchange technique is used to exchange low quantities of purifiedprotein into the detergents being used in the screening panel ofmultiple detergents. In one aspect, the technique involves binding theprotein to an affinity resin, extensively washing with a new detergent,and then eluting from the chamber or well in the new detergent.Optionally a column could be used and optionally the filtrationprocesses can be enhanced by centrifugation of the well, chamber, orplate being used. In another aspect, filtration can be enhance orspeeded up by vacuum. In parallel, aliquots of the eluates are passedthrough at least two different molecular weight cut-off filters (Low andHigh) and the amount of protein in the filtrates are measured. In oneaspect, the molecular weight cut-offs are about 100 kDa (Low) and about300 kDa (High). One of skill in the art will appreciate that the termsLow and High are relative and that the two molecular weight cut-offfilter sizes described can be modified according to the protein beingstudied or to other conditions that may effect the flow through.

One of skill in the art will appreciate that the practice of theinvention is not limited to the specific devices described herein forfiltration.

In one aspect, the amount of protein is measured by dot blot analysis ora rapid Western-blot protocol. Methods are provided herein for measuringand quantifying the dots or Western blots, such as by scanning with aninfrared imaging system to determine spot intensities, followed bysoftware analysis, normalization, and graphing. One of ordinary skill inthe art will appreciate that other methods may also be used to detectand quantify protein amounts.

In one aspect, the Differential Filtration Assay is performed using atleast one 96-well SBS format filter plate.

In one aspect, about 1000 micrograms or less of a protein of interest isneeded to perform the Differential Filtration Assay. In another aspect,about 500 micrograms or less is needed. In a further aspect, about 400micrograms or less is needed. In yet another aspect, about 200micrograms or less is needed. In a further aspect, about 50 microgramsor less is needed.

In one embodiment, the entire assay is performed in less than two hours.In one aspect, the assay is performed in less than one hour.

In one embodiment, the concentration used of a second detergent is itscritical micelle concentration.

Other detergents not described herein can also be used in the assay,either as additional detergents or to substitute for a describeddetergent.

One important aspect of the present invention is use of detergentexchange, instead of detergent dilution.

In one embodiment, the present invention provides the use of at leasttwo filter plates with different molecular-weight cutoffs (MWCO) toserve as a proxy for gel filtration (size-exclusion) chromatography. Assuch, it is a novel method for detergent (or any other buffer component)exchange. In one aspect, the present invention provides a noveldetergent panel, specifically with respect to the selection ofdetergents and/or their concentrations. Commercial detergent stocksolutions are sold as a fixed percentage (typically 10% weight/volume),while the detergent concentrations in the panel of the present inventionare a function of the critical micelle concentration (CMC) of thedetergent. In one embodiment, the present invention provides for the useof molecular weight cut off determinations coupled with screening with apanel of detergents.

Non-Detergent Buffer Exchange for Proteins

The disclosure describes the use of DFA technology, previously referredto as PASS, for screening the stability and size of integral membraneproteins as a function of detergent. The 96-well SBS format plates,including those of low and high molecular weight cut-off that comprisethe technology called DF (previously referred to as “size-exclusionchromatography mimesis” or “SEC-M”), are coupled with a “composition andconcentration” formulated panel of 94 detergents to enable the rapid and“protein-efficient” screening of behavior of a purified protein againstthis set of detergents. One of ordinary skill will realize that moredetergents can be tested by using more plates or plates with more wellsor chambers. A further embodiment of DF is its use in multiple wellformats, including, but not limited to, 96, 384, and 1536 well plates,for screening any and all buffer components required to maintainstability of proteins. These include, but are not limited to, pH; ionicstrength; osmolyte; reductant; specific chemical, element, ion,cofactor, ligand, substrate, nucleic acid or other factor; and othermacromolecular components such as proteins.

Use of DFA to Characterize Functional Stability of Membrane (or Other)Proteins

The proof-of-concept and initial demonstration of utility involved theuse of recombinant membrane proteins containing poly-histidine affinitytags, and the subsequent immobilization of these proteins on IMAC(immobilized metal-affinity chromatography) resin for use in the assay.Of course, any affinity-based immobilization can also be utilized, suchas other exogenous purification tags added to aid in proteinpurification (including, but not limited to, maltose-binding protein(MBP), glutathione-S-transferase (GST), FLAG, S-tag, strep-tag,chitin-binding domain). All of these exogenously-added tags areindependent of the actual function of the protein. However, by use ofaffinity matrices specific to a given protein's function, this functioncan be readily characterized in DFA. For example, purified protein (suchas a G-protein coupled receptor [GPCR]) can be bound to aligand-affinity matrix, where the matrix consists of a high-affinityligand attached to the solid support. The, detergent exchange isperformed, using the 94-detergent panel. If a detergent eliminatesligand binding, the protein will be eluted during the exchange step,thus being removed from the resin before elution. The assay is performedin the same way as initially described, but in order to be seen afterthe DF step, the protein must possess the proper stability, size, andfunctional integrity in order to be observed.

Use of Affinity-Immobilized Protein and DF for Compound Screening

The purified protein (for example, a GPCR) is bound to a ligand-affinityresin. A compound (or set of pooled compounds) is added to each samplein the plate. If the compound is of comparable or higher affinity to theligand on the ligand-affinity resin, some fraction of the receptor willbe eluted. The amount of eluted receptor is then detected, with thefraction of receptor eluting being related to both the affinity of thecompound(s) added, and the ability of the compounds to compete for thesame binding site as that used by the ligand on the affinity resin.

Examples

A description of examples of the invention follows. Use of the inventionis not limited to these applications. In one embodiment, the inventionconsists of two parts, the detergent panel and the DF. Both of these aredescribed below along with a protocol to carry out the assay.

Disclosed herein is a microplate-based detergent screening assay calledDFA (previously called PASS, see FIG. 1). A standard buffer exchangetechnique, successfully implemented in screening detergents suitable forextracting proteins from their membrane environment [10; 11; 12], isused to exchange microgram quantities of purified membrane protein intoeach of the detergents of our 94 detergent panel (Table 1).

Table 1 summarizes the Detergent screen. The components in the detergentplate are shown along with their locations in the plate andconcentrations used. The values in parentheses in the [Det] column arethe CMC values for each detergent. Detergents in bold were purchasedfrom Avanti Polar Lipids, italics from Bachem, underlined from EMDBiosciences, and all others from Anatrace. Detergents A3 through C9 arezwitterionic while C10 through H12 are nonionic detergents.

TABLE 1 Detergent Screen Well Abbrev. Name [Det] mM A1 No detergent(−control) A2 Empty well for current detergent (+control) A3 Z3-12ZWITTERGENT ® 3-12 8.4 (2.8) A4 Z3-14 ZWITTERGENT ® 3-14 10 (0.2) A5 DMGn-Decyl-N,N-dimethylglycine 38 (19) A6 DOMGn-Dodecyl-N,N-dimethylglycine 4.5 (1.5) A7 DAOn-Decyl-N,N-dimethylamine-N-oxide 21 (10.5) A8 UDAOn-Undecyl-N,N,-dimethylamine-N-oxide 9.6 (3.2) A9 LDAOn-Dodecyl-N,N-dimethylamine-N-oxide 3 (1) A10 C-DDFOS C-DODECAFOS ™ 44(22) A11 CF-4 CYCLOFOS ™-4 28 (14) A12 CF-5 CYCLOFOS ™-5 13.5 (4.5) B1CF-6 CYCLOFOS ™-6 8.04 (2.68) B2 CF-7 CYCLOFOS ™-7 6.2 (0.62) B3 FC-10FOS-CHOLINE ®-10 22 (11) B4 FC-11 FOS-CHOLINE ®-11 5.55 (1.85) B5 FC-12FOS-CHOLINE ®-12 4.5 (1.5) B6 FC-13 FOS-CHOLINE ®-13 7.5 (0.75) B7 FC-14FOS-CHOLINE ®-14 6 (0.12) B8 FC-I11 FOS-CHOLINE ®-ISO-11 53.2 (26.6) B9FC-I11-6U FOS-CHOLINE ®-ISO-11-6U 51.6 (25.8) B10 FC-I9FOS-CHOLINE ®-ISO-9 64 (32) B11 FC-U10-11 FOS-CHOLINE ®-UNSAT-11-10 15.5(6.2) B12 DHPC 1,2-Diheptanoyl-sn-glycero-3-phosphocholine 4.2 (1.4) C1LPC-10 LysoPC-10 20 (8) C2 LPC-12 LysoPC-12 7 (0.7) C3 FOSFEN-9FOSFEN ™-9 4.05 (1.35) C4 CHAPS CHAPS 20 (8) C5 CHAPSO CHAPSO 20 (8) C6DDMAU n-Dodecyl-N,N-(dimethylammonio)undecanoate 6.5 (0.13) C7 DDMABn-Dodecyl-N,N-(dimethylammonio)butyrate 12.9 (4.3) C8 LAPAO LAPAO 4.8(1.6) C9 TRIPAO TRIPAO 13.5 (4.5) C10 T-20 TWEEN ® 20 5.9 (0.059) C11BRIJ-35 BRIJ ®35 9.1 (0.091) C12 TX-100 TRITON ® X-100 11.5 (0.23) D1TX-114 TRITON ® X-114 10 (0.2) D2 TX-305 TRITON ® X-305 6.5 (0.65) D3TX-405 TRITON ® X-405 8.1 (0.81) D4 NID-P40[Octylphenoxy]polyethoxyethanol 15 (0.3) D5 APO8 Dimethyloctylphosphineoxide 80 (40) D6 APO9 Dimethylnonylphosphine oxide 20 (10) D7 APO10Dimethyldecylphosphine oxide 13.98 (4.66) D8 APO11Dimethylundecylphosphine oxide 3.6 (1.2) D9 APO12Dimethyldodecylphosphine oxide 5.7 (0.57) D10 C6E3 Triethylene glycolmonohexyl ether 46 (23) D11 C6E4 Tetraethylene glycol monohexyl ether 60(30) D12 C6E5 Pentaethylene glycol monohexyl ether 74 (37) E1 C7E5Pentaethylene glycol monoheptyl ether 42 (21) E2 C8E4 Tetraethyleneglycol monooctyl ether 20 (8) E3 C8E5 Pentaethylene glycol monooctylether 17.75 (7.1) E4 C8E6 Hexaethylene glycol monooctyl ether 25 (10) E5C10E5 Pentaethylene glycol monodecyl ether 8.1 (0.81) E6 C10E6Hexaethylene glycol monodecyl ether 9 (0.9) E7 C10E9Polyoxyethylene(9)decyl ether 3.9 (1.3) E8 C12E8 Octaethylene glycolmonododecyl ether 9 (0.09) E9 C12E9 Polyoxyethylene(9)dodecyl ether 5(0.05) E10 C12E10 Polyoxyethylene(10)dodecyl ether 10 (0.2) E11 C13E8Polyoxyethylene(8)tridecyl ether 10 (0.1) E12 CHAP Big CHAP 8.7 (2.9) F1CHAP-D Big CHAP, deoxy 4.2 (1.4) F2 OHES Octyl-2-hydroxyethyl-sulfoxide48.4 (24.2) F3 RDHPOS Rac-2,3-dihydroxypropyloctylsulfoxide 48.4 (24.2)F4 GX-100 Genapol ® X-100 7.5 (0.15) F5 HTGn-Heptyl-β-D-thioglucopyranoside 58 (29) F6 OGn-Octyl-β-D-glucopyranoside 36 (18) F7 NG n-Nonyl-β-D-glucopyranoside16.25 (6.5) F8 CYGLU-3 CYGLU ®-3 56 (28) F9 HECAMEG HECAMEG 39 (19.5)F10 HEGA-9 Hega ®-9 78 (39) F11 C-HEGA-10 C-Hega ®-10 70 (35) F12C-HEGA-11 C-Hega ®-11 23 (11.5) G1 CYMAL-3 CYMAL ®-3 60 (30) G2 CYMAL-4CYMAL ®-4 19 (7.6) G3 CYMAL-5 CYMAL ®-5 7.2 (2.4) G4 CYMAL-6 CYMAL ®-65.6 (0.56) G5 CYMAL-7 CYMAL ®-7 9.5 (0.19) G6 DMHM2,6-Dimethyl-4-heptyl-β-D-maltoside 55 (27.5) G7 OMn-Octyl-β-D-maltopyranoside 39 (19.5) G8 NM n-Nonyl-β-D-maltopyranoside15 (6) G9 DαM n-Decyl-α-D-maltopyranoside 4.8 (1.6) G10 DMn-Decyl-β-D-maltopyranoside 5.4 (1.8) G11 UDαMn-Undecyl-α-D-maltopyranoside 5.8 (0.58) G12 UDMn-Undecyl-β-D-maltopyranoside 5.9 (0.59) H1 ωUDMω-Undecylenyl-β-D-maltopyranoside 3.6 (1.2) H2 DDαMn-Dodecyl-α-D-maltopyranoside 7.5 (0.15) H3 DDMn-Dodecyl-β-D-maltopyranoside 8.5 (0.17) H4 TDMn-Tridecyl-β-D-maltopyranoside 1.5 (0.03) H5 OTMn-Octyl-β-D-thiomaltopyranoside 21.25 (8.5) H6 NTMn-Nonyl-β-D-thiomaltopyranoside 9.6 (3.2) H7 DTMn-Decyl-β-D-thiomaltopyranoside 9 (0.9) H8 UDTMn-Undecyl-β-D-thiomaltopyranoside 10.5 (0.21) H9 DDTMn-Dodecyl-β-D-thiomaltopyranoside 5 (0.05) H10 S-8 Sucrose8 48.8 (24.4)H11 S-10 Sucrose10 7.5 (2.5) H12 S-12 Sucrose12 15 (0.3)

Table 2 provides a summary of the common or brand names for thedetergents used and their chemical names.

TABLE 2 Detergent Chemical Names Name Chemical Name ZWITTERGENT ® 3-12n-Dodecyl-N,N-dimethyl-3-ammonio-1- propanesulfonate ZWITTERGENT ® 3-14n-Tetradecyl-N,N-dimethyl-3-ammonio-1- propanesulfonate C-DODECAFOS ™Cyclododecyl-1-phosphocholine CYCLOFOS ™-44-Cyclohexyl-1-butylphosphocholine CYCLOFOS ™-55-Cyclohexyl-1-pentylphosphocholine CYCLOFOS ™-66-Cyclohexyl-1-hexylphosphocholine CYCLOFOS ™-77-Cyclohexyl-1-heptylphosphocholine FOS-CHOLINE ®-10n-Decylphosphocholine FOS-CHOLINE ®-11 n-UndecylphosphocholineFOS-CHOLINE ®-12 n-Dodecylphosphocholine FOS-CHOLINE ®-13n-Tridecylphosphocholine FOS-CHOLINE ®-14 n-TetradecylphosphocholineFOS-CHOLINE ®-ISO-11 2,8-Dimethyl-5-nonylphosphocholineFOS-CHOLINE ®-ISO- Undecyl-6-phosphocholine 11-6U FOS-CHOLINE ®-ISO-92,6-Dimethyl-4-heptylphosphocholine FOS-CHOLINE ®-10-Undecylenyl-1-phosphocholine UNSAT-11-10 LysoPC-101-Decanoyl-2-hydroxy-sn-glycero-3- phosphocholine LysoPC-121-Lauroyl-2-hydroxy-sn-glycero-3- phosphocholine FOSFEN ™-9Nonylphenylphosphocholine CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate CHAPSO 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate LAPAO 3-Dodecylamido-N,N′-dimethylpropylamine oxide TRIPAO 3-(3 Butyl-3-phenylheptanamido)-N,N-dimethylpropan-1-amine oxide TWEEN ® 20 Polyoxyethylene(20) sorbitanemonolaurate BRIJ ®35 Polyoxyethylene lauryl ether TRITON ® X-100α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-ω-hydroxy-poly(oxy-1,2-ethanediyl), average MW 647 TRITON ® X-114α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-ω-hydroxy-poly(oxy-1,2-ethanediyl), average MW 536 TRITON ® X-305α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-ω-hydroxy-poly(oxy-1,2-ethanediyl), average MW 1526 TRITON ® X-405α-[4-(1,1,3,3-Tetramethylbutyl)phenyl]-ω-hydroxy-poly(oxy-1,2-ethanediyl), average MW 1967 Big CHAPN,N′-bis-(3-D- Gluconamidopropyl)cholamide Big CHAP, deoxyN,N′-bis-(3-D- Gluconamidopropyl)deoxycholamide Genapol ® X-100Polyoxyethylene (10) Isotridecyl Ether CYGLU ®-33-Cyclohexyl-1-propyl-β-D-glucoside HECAMEGMethyl-6-O-(N-heptylcarbamoyl)-α-D- glucopyranoside Hega ®-9Nonanoyl-N-hydroxyethylglucamide C-Hega ®-10 Cyclohexylbutanoyl-N-hydroxyethylglucamide C-Hega ®-11 Cyclohexylpentanoyl-N-hydroxyethylglucamide CYMAL ®-3 3-Cyclohexyl-1-propyl-β-D-maltosideCYMAL ®-4 4-Cyclohexyl-1-butyl-β-D-maltoside CYMAL ®-55-Cyclohexyl-1-pentyl-β-D-maltoside CYMAL ®-66-Cyclohexyl-1-hexyl-β-D-maltoside CYMAL ®-77-Cyclohexyl-1-heptyl-β-D-maltoside Sucrose8n-Octanoyl-β-D-fructofuranosyl-α-D- glucopyranoside Sucrose10α-D-Glucopyranoside, β-D-Fructofuranosyl Monodecanoate Sucrose12n-Monododecanoate-α-D-glucopyranoside, β-D-Fructofuranosyl

This simple technique involves binding the protein to an affinity resin,extensively washing with the new detergent, and finally eluting from thecolumn in the new detergent. In parallel, these eluents are each passedthrough high and low molecular weight cutoff (MWCO) filter plates. Theamounts of protein in the filtrates are measured by a rapid Western-blotprotocol. DFA is performed with 96-well SBS format filter plates, forfurther increasing speed and decreasing reagent and protein costs.

The assay disclosed herein addresses the limitations of the detergentscreening methods described above, and provides measures of bothstability and rudimentary size information. Typically, results can beobtained in approximately two hours with only few hundred micrograms ofprotein required for the assay. Furthermore, DFA can be completelyautomated if desired. To validate the assay, we present DFA data of twomembrane proteins, AqpZ and KcsA. Both of these membrane proteins havebeen crystallized and their structures initially solved by otherlaboratories.

For the purpose of this application (and corresponding to a standardempirical definition), we define the stability of a membrane protein ina given detergent to be a quantity that is inversely proportional to thefraction of PDCs that form large particles or aggregates in thatspecific detergent. If all of the protein forms large particles oraggregates (which would be present in the void volume of a suitable gelfiltration column, for example), then we would call that protein sampleunstable. If none of the protein forms large particles or aggregates(such that the protein would be seen as one or more sizing peaks in theaforementioned gel filtration column), then we would call that proteinsample stable. [Of course, stability, generally a time-dependentproperty, is not affected solely by detergent.]

Materials and Methods

Detergent Panel—

All chemicals for the detergent panel were purchased from Anatrace,Avanti Polar Lipids, Bachem, and EMD Biosciences as indicated inTable 1. 2× working stock solutions were made in ultra-pure water,dispensed into 96-well plates, heat sealed with foil tape and frozen at−20° C. until needed.

MWCO Filtered Microplate Assessment—

Stock solutions of each gel filtration MW standard (Sigma and GEHealthcare) at 2-5 mg/ml were made in PBS buffer. 30 μl of each stocksolution was added to a 0.2 μm filter plate (Part #5045, Pall Corp.) and100 kDa and 300 kDa MWCO filter plates (#T-3180-14 and #T-3180-21 ISCBioexpress, or #CMR1411 and #CMR1493-1 Seahorse Labware) and spun at2000×g for 2 min. We emphasize that these values of 100 kDa and 300 kDaMWCO are the names given to these plates by the manufacturer. As will beshown, the actual MWCOs measured are different. We will henceforth usethe nomenclature “low” and “high” for the 100 kDa and 300 kDa plates,respectively, to indicate the relative difference in the molecularweight cut-offs of the filters. The filter plate flow through along withsamples of the original stock solutions were transferred to a UVtransparent 384-well plate and Abs_(280nm) was measured on a MolecularDevices SpectraMax 384 Plus spectrophotomer with PathCheck (i.e., 1 cmpath length correction) active. The percent difference between thestocks and eluate absorbance values were used to calculate the flowthrough percent for each standard through each filter type. The errorsare the standard deviations obtained from measuring three separatesamples in each filtered microplate.

Purification of AqpZ and KcsA—

Both (His)₆-AqpZ (cys-free) and (His)₆-KcsA were over-expressed andpurified from E. coli by slight modification of published methods [13;14]. The AqpZ buffer was 20 mM Tris pH 7.4, 500 mM NaCl, 10% glycerol,and 40 mM OG. The KcsA buffer was 20 mM Tris pH 7.4, 150 mM KCl, and 1mM DDM. Both purifications were only carried out to the first IMAC stepwith Co-TALON (Clontech) resin and then desalted back into therespective buffers with PD10 columns (GE Healthcare). KcsA was furtherprocessed by digestion with chymotrypsin (Worthington Biochemical Corp.)for 4 hrs at RT with a 1:25 Chymotrypsin:KcsA ratio by mass. Thechymotrypsin was removed with immobilized benzamidine (GE Healthcare).

Detergent Stability Assay—

400 μg of AqpZ or KcsA were batch-bound to 1.5 ml of Co-TALON resin(0.27 μg protein/μl resin) along with additional buffer to a finalvolume of 7.5 ml making a 20% slurry. 50 μl of the protein-bound resinslurry (10 μl resin) was then added to the 0.2 μm filter microplateusing a multichannel pipette with the ends of the tips cut to make awider bore. The resin was pelleted in the microplate by spinning at2000×g for 2 min at 4° C. The detergent containing wash solutions wereprepared from the 2× detergent stock panel and 2× buffer stocks. 3CV(Column Volumes) of the detergent wash solution was added to each welland the microplate was spun at 2000×g for 2 min at 4° C. to remove thebuffer. This washing is repeated until 20 CV of detergent wash bufferhad been added. The elution solutions were prepared from the 2×detergent stock panel and 2× elution buffer stocks (2× wash buffercontaining 1M imidazole). The detergent exchanged protein was theneluted from the resin in 6 CV of elution solution by spinning the plateat 2000×g for 2 min at 4° C. into a PCR collection plate (Abgene). 25 μlof the eluted protein was then added to the 300 kDa (i.e., high) MWCOfilter microplate and 25 μl added to the 100 kDa (i.e., low) MWCO filterplate, the plates spun at 2000×g for 2 min at 4° C. and the eluatecollected in a PCR collection plate. The Minifold I 96-well Dot-Blotapparatus (Whatman) was used to blot the elution samples onto 0.2 μmnitrocellulose (Whatman). Due to the spot-broadening effects of somedetergents, the protein was precipitated in the blotting apparatus withtrichloroacetic acid (TCA) prior to applying the vacuum. 150 μl of 10%TCA was first added to each well of the assembled dot-blot apparatusfollowed by 10 μl of the eluate from each MWCO filter plate. Vacuum wasapplied to filter the sample through the nitrocellulose and each wellwas washed with 20 mM Tris pH 7.4, 500 mM NaCl three to four times. Themembranes were quickly washed with ultra-pure water and blocked for 10min with Odyssey blocking buffer (LI-COR Biosciences). The membraneswere probed for 10 min with an IRDye® 800CW conjugated anti-6× His Tagantibody (Rockland Immunochemicals) diluted 1:10000 in Western BreezePrimary Antibody Diluent (Invitrogen). The blots were washed withWestern Breeze Antibody Wash (Invitrogen) four times for 2 min each andthen with ultra-pure water. The blots were scanned on an OdysseyInfrared Imaging System (LI-COR Biosciences) with the intensity adjustedto avoid saturation of the spots. Integrated spot intensities weremeasured with the Odyssey software and the background for each spot wascalculated from the median value of the baseline surrounding the spot.The background-corrected integrated spot intensities were then exportedto a spreadsheet for normalization and graphing.

Gel Filtration Runs—

Detergent exchanges using larger amounts of protein (12 μg protein/μlresin) for gel filtration were carried out in 0.22 μm filter spincolumns (Millipore) and a table top centrifuge using the same detergentexchange protocol above except the protein was eluted in only 3CV and150 mM EDTA was substituted for imidazole in the elution buffer in orderto avoid imidazole fluorescence during gel filtration. 10 μl elutedprotein was loaded onto a calibrated Superdex™ 200 5/150 GL “shortcolumn” (GE Healthcare) at 0.4 ml/min. The column was equilibrated inthe exchange detergent prior to sample injection. The intrinsic proteinfluorescence (Ex280 nm/Em335 nm) was monitored using a Hitachi L-2485fluorescence detector.

Other methods not described herein but which are useful in the practiceof the invention are incorporated herein by reference in their entirety,including those International Patent Application Serial No.PCT/US2010/036562 (Wiener et al.; filed May 28, 2010).

Results

Size-Exclusion Chromatography Mimesis (i.e., DF)—

The use of multiple differing molecular weight cutoff (MWCO) filters inparallel permits acquisition of rudimentary size information on PDCs. Itis important to note that MWCO filters do not have a single sharpmolecular weight cutoff. Instead, a given filter will excludeapproximately all particles above a certain size, will passapproximately all particles below a certain size, and will let throughsome fraction of particles between these two limits. For the purposes ofDFA, consider two MWCO filter plates: high and low. The high plateexcludes particles of large size and permits some or all of theremaining particles to pass through. The retentate of the high platewill consist solely of these large particles; nearly everything elsewill be in the filtrate.

The low plate has a lower size-exclusion limit. Therefore, the retentateof the low plate will include all of the retentate present in the highplate plus additional retentate arising from the lower cutoff. Thus, inthe absence of experimental error, the fraction of a sample in thefiltrate of the low plate will always be less than or equal to thefraction of that sample in the high plate filtrate.

During the conception of the detergent screening assay, gel filtrationof the eluted samples was planned to be performed using either a genericmobile phase or detergent-specific mobile phase in conjunction with anin-line fluorescence detector to follow intrinsic protein fluorescenceof the protein of interest. It became readily apparent that this wouldnot work due to the fluorescence of a large population of the detergentsin the panel themselves (data not shown). As an alternative to gelfiltration for obtaining sizing information, or at the very least toremove any protein aggregate analogous to that present in the voidvolume of a gel filtration run, filtration through MWCO PES(polyethersulfone) filters was employed. Because MWCO PES filters arenot absolute cut-offs but instead permit a range of particle sizes topass through their membrane, SBS format MWCO filterplates from severalmanufactures were evaluated. The size distribution range for each MWCOfilterplate was measured using gel filtration MW standards. A sizingmicroplate was sought that would disallow passage of blue dextran (the2000 kDa void volume MW standard) and produce a suitable MW distributionfor estimating size. A single MWCO filtered microplate was notsufficient to satisfy both criteria because the MW permeability rangewas too narrow for the low plate and too broad for the high plate. Toovercome this deficiency, low and high MWCO microplates were pairedtogether for the assay. The MW distributions of these two MWCO filteredmicroplates along with the 0.2 μm GHP (GH Polypro, hydrophobicpolypropylene) filter plate are shown in FIG. 2.

As expected, the 0.2 μm plate allows passage of all of the gelfiltration standards, with equal and complete permeability of all ofstandards except blue dextran (partially permeable). In contrast,neither the low nor the high plates allow blue dextran to flow through,and thus prevent highly aggregated protein from passing through. Withthe use of both MWCO microplates, analysis of the high microplatefiltrate reports primarily on stability, and the ratio of the low andhigh plate filtrates reports on size. Thus, MWCO filters are used tomimic sizing data obtained sizing data obtained by gel filtrationchromatography. We call this technique DF (previously referred to as“Size-exclusion Chromatography Mimesis” or “SEC-M”).

The Detergent Panel—

The assay in its current form utilizes 96-well SBS format microplates.We utilize our 94-detergent panel (Table 1) plus negative and positivepositions A1 and A2 of the microplates. The negative control containsdetergent-free buffer, which will cause the protein to precipitate onthe resin, while the positive control is the current detergentcontaining buffer. Even though it is highly probable that the currentdetergent used for a given protein is present in the panel, the workingconcentrations of the detergent may be different. The chemical (ortrademarked names), well locations, abbreviations, workingconcentrations, and CMC values for each detergent in the panel are shownin Table 1.

All of the detergents were selected for their suitability for membraneprotein studies based upon several criteria: 1) commercial availability;2) moderate or high aqueous solubility; 3) CMC values between 0.03 and40 mM; and 4) zwitterionic or nonionic headgroups. The detergent panelis first grouped into zwitterionic and nonionic sets, further ordered bychemical class, then by chain length within each chemical class whereapplicable, and finally by CMC value. This permits the facilerecognition of patterns of stability. The detergent stock plate consistsof 2× working concentration solutions dispensed and heat sealed intosingle use microplates, for ease in formulation of the wash and elutionbuffers. Each detergent's working concentration in the panel varies withrespect to its CMC value: 2× (CMC>10 mM); 2.5× (CMC 5-10 mM); 3× (CMC1-5 mM); 10× (CMC 0.5-1 mM); 50× (CMC 0.1-0.5 mM); 100× (CMC<0.5 mM).The exception is TDM where the working concentration is 50× due tosolubility issues. The use of several CMC multiples is necessary due tolow-CMC detergents being required at higher concentrations (in terms oftheir CMC) than high-CMC detergents [5].

Visualization and Quantification of the Elutions—

Using a fast Western protocol, the elutions for each MWCO microplate areblotted to a nitrocellulose membrane, visualized using a conjugatedprimary antibody specific to the affinity tag used, and quantified inapproximately one hour. Dot blots of the elutions from the AqpZ and KcsAdetergent exchanges are shown in FIG. 3. Quantification of dots on theblots measures the amount of protein eluted from each well. Because thesame amount of protein was used in each well and an incompatibledetergent will cause a membrane protein to precipitate on the affinityresin, the integrated dot intensity is directly related to the abilityof a particular detergent to stabilize a membrane protein relative tothe other detergents in the panel. As mentioned in the introduction, theloss of membrane protein stability is assayed by the presence of andamount of irreversible aggregates (large particle formation) as afunction of the detergent species that is present in the PDC. The lowand high dot blots are quantified and normalized to the highestintensity dot on each respective blot. The ratios of the normalized lowto high dot intensities are calculated to indicate the relative size ofthe PDC; this ratio is inversely proportional to the PDC size, withlarger ratio values indicative of a smaller PDC size. The ratio is abetter estimate of size than just the low dot intensities alone. The lowdot intensities are related to both size and stability, while thelow/high ratio accounts for the removal of any aggregated proteinretained by the high microplate (and thus also retained by the lowmicroplate).

FIG. 4 and FIG. 5 demonstrate two representations of the quantifieddata. FIG. 4, the “audio equalizer representation”, presents the data toallow any stability and/or size patterns related to detergent type,chain length, or CMC to be easily elucidated because the detergent panelis organized in that manner. FIG. 5, the “size-stability quad-plot”,displays the data to allow quick assessment of PDC size related tostability without any regard for the detergent's physical or chemicalproperties. The data in both representations are binned into quartilesto help compensate for the inherent error in measuring only one datapoint. Interestingly, despite AqpZ being a larger tetramer (103 kDa),the KcsA tetramer (55 kDa) possesses a much more variable sizedependence as demonstrated by the broader distribution of relative PDCsize seen in FIG. 5.

DF Correlates with Gel Filtration—

In order to further validate the use of the DF technique in the assay,larger scale detergent exchanges were performed on AqpZ and KcsA andsamples were run over a calibrated gel filtration column. The retentiontimes of the gel filtration runs were compared to the dot intensitiesobtained from the two MWCO filter plate elutions. FIG. 6 (top panel)shows three AqpZ gel filtration runs exchanged into TDM, LDAO, and OGalong with the corresponding dot blots for those detergents. Aspredicted from the low elution dot blots, the apparent PDC size order ofAqpZ is OG<LDAO<TDM which is inversely proportional to their respectivedot blot intensities.

FIG. 6 (bottom panel) demonstrates an interesting phenomenon in whichthe same detergent at two different concentrations gives two differentPDC sizes for KcsA. KcsA in the traditional working concentration of 1mM DDM is predicted to have a larger PDC size than the same protein in8.5 mM DDM by both gel filtration and DF. The reason for a higherconcentration of detergent reducing the size of the PDC is currentlyunder investigation.

Discussion

We have developed a microplate-based detergent screening assay, theDifferential Filtration Assay (DFA). Differential filtration (DF), thesimple underlying principle, is that the variation of amounts ofmacromolecules in filtrates obtained by passage of a macromolecularsolution through several different molecular weight cutoff (MWCO)filters provides information on the stability (aggregation) and size ofa macromolecular solute as a function of buffer composition. DF rapidlycaptures significant information that is typically obtained, much moreslowly, by SEC. The combination of DF with a specific method fordetection and quantitation of filtrates yields a Differential FiltrationAssay (DFA). For membrane proteins in PDCs, the detergent of the PDC isoften the most significant buffer component; however, DF is equallywell-suited for examination of any solution conditions for anymacromolecular solute.

Differential Filtration (DF)

The use of multiple differing molecular weight cutoff (MWCO) filters inparallel permits acquisition of rudimentary size information on PDCs. Itis important to note that MWCO filters do not have a single sharpmolecular weight cutoff Instead, a given filter will excludeapproximately all particles above a certain size, will passapproximately all particles below a certain size, and will let throughsome fraction of particles between these two limits. For the purposes ofDFA, consider two MWCO filter plates: high and low. The high plateexcludes particles of large size and permits some or all of theremaining particles to pass through. The retentate of the high platewill consist solely of these large particles; nearly everything elsewill be in the filtrate. The low plate has a lower size-exclusion limit.Therefore, the retentate of the low plate will include all of theretentate present in the high plate plus additional retentate arisingfrom the lower cutoff. Thus, in the absence of experimental error, thefraction of a sample in the filtrate of the low plate will always beless than or equal to the fraction of that sample in the high platefiltrate.

As an alternative to gel filtration for obtaining sizing information, orat the very least to remove any protein aggregate analogous to thatpresent in the void volume of a gel filtration run, filtration throughMWCO PES (polyethersulfone) filters was employed. Since MWCO PES filtersare not absolute cutoffs but instead permit a range of particle sizes topass through their membrane, SBS format MWCO filterplates from severalmanufactures were evaluated. The size distribution range for each MWCOfilterplate was measured using gel filtration MW standards. A sizingmicroplate was sought that would disallow passage of blue dextran (the2000 kDa void volume MW standard) and produce a suitable MW distributionfor estimating size. We emphasize that a single MWCO filtered microplatewas not sufficient to satisfy both criteria since the MW permeabilityrange was too narrow for the low plate and too broad for the high plate.To overcome this deficiency, low and high MWCO microplates were pairedtogether for the assay. With the use of both MWCO microplates, analysisof the high microplate filtrate reports primarily on stability, and theratio of the low and high plate filtrates reports on size. Thus,analysis of the filtrates of several different MWCO filters capturessignificant data that is typically obtained by SEC. We call thistechnique Differential Filtration (DF).

The detergent panel presented here, combined with DFA, provides a robustand rapid means to survey membrane protein stability in a large numberof chemically diverse detergents as well as to obtain rudimentary PDCsizing information. While the sizing information obtained from DF doesnot provide an absolute value for apparent MW, it does provide a coarsefilter that allows the results to be binned and ranked for furtheranalysis via traditional SEC as desired. The ability to obtain quicklythis sizing and stability information is a significant benefit to thismethod, especially if the time that would be required to examine all 94samples with SEC is considered. For the Superdex™ 200 5/150 GL columnused in this work, each run is approximately 6 minutes in duration. Useof a generic mobile phase would require approximately 10 hrs, and use ofdetergent-specific mobile phases would require an additional 44 hrs forcolumn equilibration and pump washing. These time estimates assume nodown time and complete automation of the chromatography. Furthermore,many protein samples will aggregate or denature during this time,leading to ambiguous results.

The core technology of the Differential Filtration Assay (DFA) is theuse of several different MWCO filters which yield differentialfiltration (DF) of a macromolecular solute. Detection and quantificationof the filtrates, required for application of DF to this specific assay,can be performed by a variety of methods. Factors that influence thechoice of detection method include: specific vs. non-specific detection,direct vs. indirect detection, sensitivity, accuracy, precision, timerequired, and (of course) amount of protein required. Specific detectionmethods are those that will, essentially, detect only the expressedprotein of interest. In this publication, we performed a rapid Westernblot protocol which utilized a fluorescent primary antibody to thepolyhistidine affinity tag. Antibody-based methods are highly specific,and indirect. A benefit of this high specificity is the ability toperform DFA on less highly-purified samples. However, measurement ofantibody binding to quantitate antigen, an indirect method, canintroduce significant error, especially if the measurement is performedonly once (illustrated by the analysis of error that we have presentedhere). While error can be decreased by performing multiple measurementsof each sample-buffer condition, this increases the time, cost, andamount of protein required to perform the assay. A detection method,that is specific and direct, is measurement of the fluorescence of asmall-molecule fluorophore (such as FlAsH) attached to agenetically-encoded binding site of the recombinant protein, or of afluorescent protein (such as GFP) made as a fusion protein with thetarget. These are also direct detection methods, as the protein itselfis detected. Non-specific direct detection methods are those whichdetect total amount of protein in the filtrate. These include Lowry andBCA assays, as well as non-specific fluorescent labeling of proteins(such as by amine-reactive fluorophores). A likely advantage of directfluorescent methods, whether specific or non-specific, is that the highand low MWCO filtrates can go directly into fluorescence microplates fordirect reading in a fluorescence plate reader.

With structural biology methodologies moving towards performingexperiments on smaller scales with smaller amounts of material(especially important for difficult research problems with limitedand/or expensive reagents), the development and use of high throughputmethodologies have increased. We present here a true high throughputmembrane protein detergent screening assay that can be completed inapproximately two hours with microgram amounts of protein and microlitervolumes of reagents. DFA helps to overcome the barrier of low proteinyields which are unfortunately typical for membrane proteins, especiallywhen using more complex and/or higher order expression systems (e.g.eukaryotic). The assay as presented here used 400 μg of protein toobtain stability and PDC sizing information on 94 different detergentsfrom our panel. This amount of protein is not the absolute minimumamount required to perform the assay. DFA could be conducted with 10-50μg of protein (or even less) if more sensitive detection of the low andhigh MWCO elutions could be performed. While we have focused solely uponthe use of DFA for the parallel screening of multiple single detergents,DFA can readily be extended to the screening of detergent mixtures,additives, ionic strength, pH and any other variable buffer componentsfor both membrane and soluble proteins. Lastly, for proteins thatpossess native in vivo ligand-binding function, use of “physiological”ligand-binding affinity matrices in DFA can provide functional, as wellas stability and size, characterization.

Upon inspection of FIG. 4, various detergent stability and size trendsare seen for AqpZ and KcsA. We present several examples of the types ofobservations that can be obtained from DFA. For AqpZ, there is increasedstability with an increase in detergent chain length seen withdimethylamine-N-oxide detergents (A7-A9), while decreased stability isobserved with increasing ethylene chain length in the C8En detergents(E2-E4). For KcsA, increasing detergent chain length for glucosidedetergents (F5-F7) resulted in decreased stability while just theaddition of a hydroxyl group on Big CHAP (E12) forming Big CHAP, deoxy(F1) increases protein stability.

As mentioned in Results, FIG. 6 shows an interesting phenomenon in whichthe smaller KcsA tetramer (55 kDa) has a broader size distribution thanthat of the larger AqpZ tetramer (103 kDa). This is most likely relatedto the amount of detergent KcsA binds relative to AqpZ to maintain itssolubility (i.e. the more bound detergent, the larger the PDC size). Theobservation that a higher concentration of DDM resulted in smaller PDCsize for KcsA (FIG. 6, bottom panel) suggests that more detergent isrequired to keep KcsA in a more compact state. Because the DDM monomerconcentration should be relatively constant and approximately equal tothe CMC at both 1 mM and 8.5 mM DDM concentrations [15], a higher numberof DDM micelles appears to be required to form a smaller PDC.Experiments, planned to investigate this phenomenon, includedetermination of the amount of DDM bound to KcsA as a function of DDMconcentration. The amount of detergent present in the PDC can bedetermined by static light-scattering coupled with refractive index andUV detection [16; 17; 18; 19]. In the context of the results observedwith KcsA, we note that, contrary to the most simple expectations,detergent micelle size can change as a function of detergentconcentration [20; 21].

The readout from the size-stability quad-plot (e.g., FIG. 5) may helppredict which detergents are best for crystallization because eachquadrant represents different levels of stability and PDC size. Futureexperiments are planned to determine if a “crystallization-quadrant”exists. This would be somewhat analogous to the second virialcoefficient (B₂₂) “crystallization slot” where it was demonstrated thatsoluble proteins with B₂₂ values within a specific narrow range had apropensity to form crystals [22]. Similar observations have beenextended to membrane proteins [23; 24; 25; 26]. While detergents in thesmallest, most stable quadrant would likely be useful for NMR, there isnot enough data at present to suggest that this is necessarily the bestcriteria for membrane protein crystallography. Whether or not acrystallization quadrant exists, our assay and detergent panel will helpwith the critical “pipeline step” of choosing the proper detergents forany membrane protein of interest quickly and with minimal quantities ofreagents.

The two test proteins, AqpZ and KcsA, used in this study have both hadtheir structures determined [13; 14; 27]. What is the location in thesize-stability quad-plot for the “crystallization” detergents of AqpZand KcsA? AqpZ was crystallized in n-octyl-β-D-glucopyranoside (OG) [14;28]. OG [F6, Table 1] is located in the second-highest stability andsecond-smallest size quadrant (FIG. 5). For KcsA, Doyle et al. reportpurification of KcsA in n-decyl-β-D-maltopyranoside (DM) followed bydetergent exchange via dialysis into n-dodecyl-N,N-dimethylamine-N-oxide(LDAO) [13]. DM [G10, Table 1] is located in the second-higheststability and second-smallest size quadrant (FIG. 5). LDAO [A9, Table 1]is located in the third-highest (second-lowest) stability and smallestsize quadrant (FIG. 5). As the degree of exchange is not stated, wecannot unambiguously assign accurately the “crystallization” detergent(or detergent mixture) of KcsA. However, it is interesting to note thatcrystal structures of KcsA:Fab complexes (e.g., [29]) have essentiallyall been in DM. Based upon these scant examples, it is tempting tospeculate that crystallization may be best pursued from the upper-left“quarter” of the quad-plot (i.e., those detergents that lay within themore stable and smaller halves of the distribution), but suchconclusions await further data.

Compared to commercial detergent panels, the detergent panel containsonly those detergents that are considered non-denaturing and atconcentrations that account for the varying CMC values. Furthermore, apanel as described herein is expanded compared to commercial detergentscreens.

BIBLIOGRAPHY

-   [1] E. Granseth, D. O. Daley, M. Rapp, K. Melen, and G. von Heijne,    Experimentally constrained topology models for 51,208 bacterial    inner membrane proteins. J Mol Biol 352 (2005) 489-94.-   [2] A. Krogh, B. Larsson, G. von Heijne, and E. L. Sonnhammer,    Predicting transmembrane protein topology with a hidden Markov    model: application to complete genomes. J Mol Biol 305 (2001)    567-80.-   [3] E. Wallin, and G. von Heijne, Genome-wide analysis of integral    membrane proteins from eubacterial, archaean, and eukaryotic    organisms. Protein Sci 7 (1998) 1029-38.-   [4] M. C. Wiener, Existing and emergent roles for surfactants in the    three-dimensional crystallization of integral membrane proteins.    Current Opinion in Colloid & Interface Science 6 (2001) 412-419.-   [5] M. C. Wiener, A pedestrian guide to membrane protein    crystallization. Methods 34 (2004) 364-72.-   [6] P. Raman, V. Cherezov, and M. Caffrey, The Membrane Protein Data    Bank. Cell Mol Life Sci 63 (2006) 36-51.-   [7] S. Newstead, S. Ferrandon, and S. Iwata, Rationalizing α-helical    membrane protein crystallization. Protein Sci 17 (2008) 466-472.-   [8] D. A. Gutmann, E. Mizohata, S. Newstead, S. Ferrandon, V.    Postis, X. Xia, P. J. Henderson, H. W. van Veen, and B. Byrne, A    high-throughput method for membrane protein solubility screening:    the ultracentrifugation dispersity sedimentation assay. Protein Sci    16 (2007) 1422-8.-   [9] Kawate et al., Fluorescence-detection size-exclusion    chromatography for precrystallization screening of integral membrane    proteins. Structure 14 (2006) 673-81.-   [10] G. E. Healthcare Data File: His MultiTrapFF and His Multitrap    HP. 11-0036-63 AB (2007).-   [11] S. Eshaghi, High-throughput expression and detergent screening    of integral membrane proteins. Methods Mol Biol 498 (2009) 265-71.-   [12] D. Niegowski, M. Hedren, P. Nordlund, and S. Eshaghi, A simple    strategy towards membrane protein purification and crystallization.    Int J Biol Macromol 39 (2006) 83-7.-   [13] D. A. Doyle, J. Morais Cabral, R. A. Pfuetzner, A. Kuo, J. M.    Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon, The structure of    the potassium channel: molecular basis of K+ conduction and    selectivity. Science 280 (1998) 69-77.-   [14] D. F. Savage, P. F. Egea, Y. Robles-Colmenares, J. D.    O'Connell, 3rd, and R. M.-   Stroud, Architecture and selectivity in aquaporins: 2.5 a X-ray    structure of aquaporin Z. PLoS Biol 1 (2003) E72.-   [15] M. Zulauf, Detergent phenomena in membrane protein    crystallization. in: H. Michel, (Ed.), Crystallization of Membrane    Proteins, CRC Press, Boca Raton, Fla., 1991, pp. 53-72.-   [16] R. A. Albright, J. L. Ibar, C. U. Kim, S. M. Gruner, and J. H.    Morais-Cabral, The RCK domain of the KtrAB K+transporter: multiple    conformations of an octameric ring. Cell 126 (2006) 1147-59.-   [17] Y. Hayashi, H. Matsui, and T. Takagi, Membrane protein    molecular weight determined by low-angle laser light-scattering    photometry coupled with high-performance gel chromatography. Methods    Enzymol 172 (1989) 514-28.-   [18] J. F. White, J. Grodnitzky, J. M. Louis, L. B. Trinh, J.    Shiloach, J. Gutierrez, J. K. Northup, and R. Grisshammer,    Dimerization of the class A G protein-coupled neurotensin receptor    NTS1 alters G protein interaction. Proc Natl Acad Sci USA 104 (2007)    12199-204.-   [19] D. Yernool, O. Boudker, E. Folta-Stogniew, and E. Gouaux,    Trimeric subunit stoichiometry of the glutamate transporters from    Bacillus caldotenax and Bacillus stearothermophilus. Biochemistry    42 (2003) 12981-8.-   [20] P. G. Nilsson, H. Wennerstrom, and B. Lindman, Structure of    micellar solutions of non-ionic surfactants—nuclear    magnetic-resonance self-diffusion and proton relaxation studies of    poly(ethylene oxide) alkyl ethers. J Phys Chem 87 (1983) 1377-1385.-   [21] R. W. Roxby, and B. P. Mills, Micelle size distribution and    free monomer concentration in aqueous-solutions of octyl glucoside.    J Phys Chem 94 (1990) 456-459.-   [22] A. George, and W. W. Wilson, Predicting protein crystallization    from a dilute solution property. Acta Crystallogr D Biol Crystallogr    50 (1994) 361-5.-   [23] P. J. Loll, M. Allaman, and J. Wiencek, Assessing the role of    detergent-detergent interactions in membrane protein    crystallization. J. Crystal Growth 232 (2001) 1-4.-   [24]C. Hitscherich, J. Kaplan, M. Allaman, J. Wiencek, and P. J.    Loll, Static light scattering studies of OmpF porin: implications    for integral membrane proteins. Protein Sci 9 (2000) 1559-1566.-   [25] B. W. Berger, C. M. Gendron, A. M. Lenhoff, and E. W. Kaler,    Effects of additives on surfactant phase behavior relevant to    bacteriorhodopsin crystallization. Protein Sci 15 (2006) 2682-2696.-   [26]M. C. Wiener, When worlds colloid. Protein Sci 15 (2006)    2679-2681.-   [27] J. Jiang, B. V. Daniels, and D. Fu, Crystal structure of AqpZ    tetramer reveals two distinct Arg-189 conformations associated with    water permeation through the narrowest constriction of the    water-conducting channel. J Biol Chem 281 (2006) 454-60.-   [28] B. V. Daniels, J. S. Jiang, and D. Fu, Crystallization and    preliminary crystallographic analysis of the Escherichia coli water    channel AqpZ. Acta Crystallogr D Biol Crystallogr 60 (2004) 561-3.-   [29] Y. F. Zhou, J. H. Morais-Cabral, A. Kaufman, and R. MacKinnon,    Chemistry of ion coordination and hydration revealed by a    K+channel-Fab complex at 2.0 angstrom resolution. Nature 414 (2001)    43-48.-   30. Postis et al., Mol Membr Biol. 2008 December; 25(8):617-24.-   31. Tate, Practical considerations of membrane protein instability    during purification and crystallisation, Methods Mol Biol. 2010;    601:187-203.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

What is claimed is:
 1. A method for determining the stability and sizeof a protein in a detergent, said method comprising: a) obtaining asolution comprising said protein in a first detergent; b) adding aneffective amount of an affinity resin to said solution; c) adding analiquot of said solution comprising said protein in a first detergentand an affinity resin to a first chamber, said first chamber comprisinga filter wherein said pore size is about 0.2 μm; d) washing said firstchamber with a wash solution comprising a different detergent; e)eluting said protein in said first chamber with an elution solutioncomprising said different detergent and collecting the eluate; f)passing an aliquot of said eluate through a second chamber comprising ahigh molecular weight cut-off filter and another aliquot of said eluatethrough a third chamber comprising a low molecular weight cut-offfilter; g) measuring the amount of protein in the eluate passing throughthe high molecular weight cut-off filter and the amount of protein inthe eluate passing through the low molecular weight cut-off filter; andh) comparing the amount of protein eluted through the high molecularweight cut-off filter with the amount of protein eluted through the lowmolecular weight cut-off filter, thereby determining the stability andsize of a protein in at least one detergent.
 2. The method of claim 1,wherein said first chamber is a well of a multiwell microplate.
 3. Themethod of claim 1, wherein said second chamber is a well of a multiwellmicroplate, further wherein each well comprises a high molecular weightcut-off filter.
 4. The method of claim 1, wherein said third chamber isa well of a multiwell microplate, further wherein each well comprises alow molecular weight cut-off filter.
 5. The method of claim 2, whereinat least two wells are used.
 6. The method of claim 5, wherein multipledifferent detergents are used.
 7. The method of claim 6, wherein whenmultiple detergents are tested, each well comprising a detergentcomprises a different detergent than the other wells comprising adetergent, and optionally one or more wells comprise a positive controland one or more wells comprise a negative control.
 8. The method ofclaim 1, wherein about 20 column volumes of said wash solution are usedfor said wash.
 9. The method of claim 1, wherein about 6 column volumesof said elution solution are used for said elution.
 10. The method ofclaim 1, wherein said high molecular weight cut-off filter is about 300kDa.
 11. The method of claim 1, wherein said low molecular weightcut-off filter is about 100 kDa.
 12. The method of claim 7, wherein saidmultiwell plate is a 96, 384, or 1536 well plate.
 13. The method ofclaim 12, wherein said 96 well plate is a Society for BiomolecularSciences format plate.
 14. The method of claim 12, wherein 94 differentdetergents are tested.
 15. The method of claim 1, wherein said differentdetergent is selected from the following table: Well Abbrev. Name [Det]mM A1 No detergent (−control) A2 Empty well for current detergent(+control) A3 Z3-12 ZWITTERGENT ® 3-12 8.4 (2.8) A4 Z3-14 ZWITTERGENT ®3-14 10 (0.2) A5 DMG n-Decyl-N,N-dimethylglycine 38 (19) A6 DOMGn-Dodecyl-N,N-dimethylglycine 4.5 (1.5) A7 DAOn-Decyl-N,N-dimethylamine-N-oxide 21 (10.5) A8 UDAOn-Undecyl-N,N,-dimethylamine-N-oxide 9.6 (3.2) A9 LDAOn-Dodecyl-N,N-dimethylamine-N-oxide 3 (1) A10 C-DDFOS C-DODECAFOS ™ 44(22) A11 CF-4 CYCLOFOS ™-4 28 (14) A12 CF-5 CYCLOFOS ™-5 13.5 (4.5) B1CF-6 CYCLOFOS ™-6 8.04 (2.68) B2 CF-7 CYCLOFOS ™-7 6.2 (0.62) B3 FC-10FOS-CHOLINE ®-10 22 (11) B4 FC-11 FOS-CHOLINE ®-11 5.55 (1.85) B5 FC-12FOS-CHOLINE ®-12 4.5 (1.5) B6 FC-13 FOS-CHOLINE ®-13 7.5 (0.75) B7 FC-14FOS-CHOLINE ®-14 6 (0.12) B8 FC-I11 FOS-CHOLINE ®-ISO-11 53.2 (26.6) B9FC-I11-6U FOS-CHOLINE ®-ISO-11-6U 51.6 (25.8) B10 FC-I9FOS-CHOLINE ®-ISO-9 64 (32) B11 FC-U10-11 FOS-CHOLINE ®-UNSAT-11-10 15.5(6.2) B12 DHPC 1,2-Diheptanoyl-sn-glycero-3-phosphocholine 4.2 (1.4) C1LPC-10 LysoPC-10 20 (8) C2 LPC-12 LysoPC-12 7 (0.7) C3 FOSFEN-9FOSFEN ™-9 4.05 (1.35) C4 CHAPS CHAPS 20 (8) C5 CHAPSO CHAPSO 20 (8) C6DDMAU n-Dodecyl-N,N-(dimethylammonio)undecanoate 6.5 (0.13) C7 DDMABn-Dodecyl-N,N-(dimethylammonio)butyrate 12.9 (4.3) C8 LAPAO LAPAO 4.8(1.6) C9 TRIPAO TRIPAO 13.5 (4.5) C10 T-20 TWEEN ® 20 5.9 (0.059) C11BRIJ-35 BRIJ ®35 9.1 (0.091) C12 TX-100 TRITON ® X-100 11.5 (0.23) D1TX-114 TRITON ® X-114 10 (0.2) D2 TX-305 TRITON ® X-305 6.5 (0.65) D3TX-405 TRITON ® X-405 8.1 (0.81) D4 NID-P40[Octylphenoxy]polyethoxyethanol 15 (0.3) D5 APO8 Dimethyloctylphosphineoxide 80 (40) D6 APO9 Dimethylnonylphosphine oxide 20 (10) D7 APO10Dimethyldecylphosphine oxide 13.98 (4.66) D8 APO11Dimethylundecylphosphine oxide 3.6 (1.2) D9 APO12Dimethyldodecylphosphine oxide 5.7 (0.57) D10 C6E3 Triethylene glycolmonohexyl ether 46 (23) D11 C6E4 Tetraethylene glycol monohexyl ether 60(30) D12 C6E5 Pentaethylene glycol monohexyl ether 74 (37) E1 C7E5Pentaethylene glycol monoheptyl ether 42 (21) E2 C8E4 Tetraethyleneglycol monooctyl ether 20 (8) E3 C8E5 Pentaethylene glycol monooctylether 17.75 (7.1) E4 C8E6 Hexaethylene glycol monooctyl ether 25 (10) E5C10E5 Pentaethylene glycol monodecyl ether 8.1 (0.81) E6 C10E6Hexaethylene glycol monodecyl ether 9 (0.9) E7 C10E9Polyoxyethylene(9)decyl ether 3.9 (1.3) E8 C12E8 Octaethylene glycolmonododecyl ether 9 (0.09) E9 C12E9 Polyoxyethylene(9)dodecyl ether 5(0.05) E10 C12E10 Polyoxyethylene(10)dodecyl ether 10 (0.2) E11 C13E8Polyoxyethylene(8)tridecyl ether 10 (0.1) E12 CHAP Big CHAP 8.7 (2.9) F1CHAP-D Big CHAP, deoxy 4.2 (1.4) F2 OHES Octyl-2-hydroxyethyl-sulfoxide48.4 (24.2) F3 RDHPOS Rac-2,3-dihydroxypropyloctylsulfoxide 48.4 (24.2)F4 GX-100 Genapol ® X-100 7.5 (0.15) F5 HTGn-Heptyl-β-D-thioglucopyranoside 58 (29) F6 OGn-Octyl-β-D-glucopyranoside 36 (18) F7 NG n-Nonyl-β-D-glucopyranoside16.25 (6.5) F8 CYGLU-3 CYGLU ®-3 56 (28) F9 HECAMEG HECAMEG 39 (19.5)F10 HEGA-9 Hega ®-9 78 (39) F11 C-HEGA-10 C-Hega ®-10 70 (35) F12C-HEGA-11 C-Hega ®-11 23 (11.5) G1 CYMAL-3 CYMAL ®-3 60 (30) G2 CYMAL-4CYMAL ®-4 19 (7.6) G3 CYMAL-5 CYMAL ®-5 7.2 (2.4) G4 CYMAL-6 CYMAL ®-65.6 (0.56) G5 CYMAL-7 CYMAL ®-7 9.5 (0.19) G6 DMHM2,6-Dimethyl-4-heptyl-β-D-maltoside 55 (27.5) G7 OMn-Octyl-β-D-maltopyranoside 39 (19.5) G8 NM n-Nonyl-β-D-maltopyranoside15 (6) G9 DαM n-Decyl-α-D-maltopyranoside 4.8 (1.6) G10 DMn-Decyl-β-D-maltopyranoside 5.4 (1.8) G11 UDαMn-Undecyl-α-D-maltopyranoside 5.8 (0.58) G12 UDMn-Undecyl-β-D-maltopyranoside 5.9 (0.59) H1 ωUDMω-Undecylenyl-β-D-maltopyranoside 3.6 (1.2) H2 DDαMn-Dodecyl-α-D-maltopyranoside 7.5 (0.15) H3 DDMn-Dodecyl-β-D-maltopyranoside 8.5 (0.17) H4 TDMn-Tridecyl-β-D-maltopyranoside 1.5 (0.03) H5 OTMn-Octyl-β-D-thiomaltopyranoside 21.25 (8.5) H6 NTMn-Nonyl-β-D-thiomaltopyranoside 9.6 (3.2) H7 DTMn-Decyl-β-D-thiomaltopyranoside 9 (0.9) H8 UDTMn-Undecyl-β-D-thiomaltopyranoside 10.5 (0.21) H9 DDTMn-Dodecyl-β-D-thiomaltopyranoside 5 (0.05) H10 S-8 Sucrose8 48.8 (24.4)H11 S-10 Sucrose10 7.5 (2.5) H12 S-12 Sucrose12 15 (0.3)


16. The method of claim 14, wherein said detergents and controls areselected from the following table: Well Abbrev. Name [Det] mM A1 Nodetergent (−control) A2 Empty well for current detergent (+control) A3Z3-12 ZWITTERGENT ® 3-12 8.4 (2.8) A4 Z3-14 ZWITTERGENT ® 3-14 10 (0.2)A5 DMG n-Decyl-N,N-dimethylglycine 38 (19) A6 DOMGn-Dodecyl-N,N-dimethylglycine 4.5 (1.5) A7 DAOn-Decyl-N,N-dimethylamine-N-oxide 21 (10.5) A8 UDAOn-Undecyl-N,N,-dimethylamine-N-oxide 9.6 (3.2) A9 LDAOn-Dodecyl-N,N-dimethylamine-N-oxide 3 (1) A10 C-DDFOS C-DODECAFOS ™ 44(22) A11 CF-4 CYCLOFOS ™-4 28 (14) A12 CF-5 CYCLOFOS ™-5 13.5 (4.5) B1CF-6 CYCLOFOS ™-6 8.04 (2.68) B2 CF-7 CYCLOFOS ™-7 6.2 (0.62) B3 FC-10FOS-CHOLINE ®-10 22 (11) B4 FC-11 FOS-CHOLINE ®-11 5.55 (1.85) B5 FC-12FOS-CHOLINE ®-12 4.5 (1.5) B6 FC-13 FOS-CHOLINE ®-13 7.5 (0.75) B7 FC-14FOS-CHOLINE ®-14 6 (0.12) B8 FC-I11 FOS-CHOLINE ®-ISO-11 53.2 (26.6) B9FC-I11-6U FOS-CHOLINE ®-ISO-11-6U 51.6 (25.8) B10 FC-I9FOS-CHOLINE ®-ISO-9 64 (32) B11 FC-U10-11 FOS-CHOLINE ®-UNSAT-11-10 15.5(6.2) B12 DHPC 1,2-Diheptanoyl-sn-glycero-3-phosphocholine 4.2 (1.4) C1LPC-10 LysoPC-10 20 (8) C2 LPC-12 LysoPC-12 7 (0.7) C3 FOSFEN-9FOSFEN ™-9 4.05 (1.35) C4 CHAPS CHAPS 20 (8) C5 CHAPSO CHAPSO 20 (8) C6DDMAU n-Dodecyl-N,N-(dimethylammonio)undecanoate 6.5 (0.13) C7 DDMABn-Dodecyl-N,N-(dimethylammonio)butyrate 12.9 (4.3) C8 LAPAO LAPAO 4.8(1.6) C9 TRIPAO TRIPAO 13.5 (4.5) C10 T-20 TWEEN ® 20 5.9 (0.059) C11BRIJ-35 BRIJ ®35 9.1 (0.091) C12 TX-100 TRITON ® X-100 11.5 (0.23) D1TX-114 TRITON ® X-114 10 (0.2) D2 TX-305 TRITON ® X-305 6.5 (0.65) D3TX-405 TRITON ® X-405 8.1 (0.81) D4 NID-P40[Octylphenoxy]polyethoxyethanol 15 (0.3) D5 APO8 Dimethyloctylphosphineoxide 80 (40) D6 APO9 Dimethylnonylphosphine oxide 20 (10) D7 APO10Dimethyldecylphosphine oxide 13.98 (4.66) D8 APO11Dimethylundecylphosphine oxide 3.6 (1.2) D9 APO12Dimethyldodecylphosphine oxide 5.7 (0.57) D10 C6E3 Triethylene glycolmonohexyl ether 46 (23) D11 C6E4 Tetraethylene glycol monohexyl ether 60(30) D12 C6E5 Pentaethylene glycol monohexyl ether 74 (37) E1 C7E5Pentaethylene glycol monoheptyl ether 42 (21) E2 C8E4 Tetraethyleneglycol monooctyl ether 20 (8) E3 C8E5 Pentaethylene glycol monooctylether 17.75 (7.1) E4 C8E6 Hexaethylene glycol monooctyl ether 25 (10) E5C10E5 Pentaethylene glycol monodecyl ether 8.1 (0.81) E6 C10E6Hexaethylene glycol monodecyl ether 9 (0.9) E7 C10E9Polyoxyethylene(9)decyl ether 3.9 (1.3) E8 C12E8 Octaethylene glycolmonododecyl ether 9 (0.09) E9 C12E9 Polyoxyethylene(9)dodecyl ether 5(0.05) E10 C12E10 Polyoxyethylene(10)dodecyl ether 10 (0.2) E11 C13E8Polyoxyethylene(8)tridecyl ether 10 (0.1) E12 CHAP Big CHAP 8.7 (2.9) F1CHAP-D Big CHAP, deoxy 4.2 (1.4) F2 OHES Octyl-2-hydroxyethyl-sulfoxide48.4 (24.2) F3 RDHPOS Rac-2,3-dihydroxypropyloctylsulfoxide 48.4 (24.2)F4 GX-100 Genapol ® X-100 7.5 (0.15) F5 HTGn-Heptyl-β-D-thioglucopyranoside 58 (29) F6 OGn-Octyl-β-D-glucopyranoside 36 (18) F7 NG n-Nonyl-β-D-glucopyranoside16.25 (6.5) F8 CYGLU-3 CYGLU ®-3 56 (28) F9 HECAMEG HECAMEG 39 (19.5)F10 HEGA-9 Hega ®-9 78 (39) F11 C-HEGA-10 C-Hega ®-10 70 (35) F12C-HEGA-11 C-Hega ®-11 23 (11.5) G1 CYMAL-3 CYMAL ®-3 60 (30) G2 CYMAL-4CYMAL ®-4 19 (7.6) G3 CYMAL-5 CYMAL ®-5 7.2 (2.4) G4 CYMAL-6 CYMAL ®-65.6 (0.56) G5 CYMAL-7 CYMAL ®-7 9.5 (0.19) G6 DMHM2,6-Dimethyl-4-heptyl-β-D-maltoside 55 (27.5) G7 OMn-Octyl-β-D-maltopyranoside 39 (19.5) G8 NM n-Nonyl-β-D-maltopyranoside15 (6) G9 DαM n-Decyl-α-D-maltopyranoside 4.8 (1.6) G10 DMn-Decyl-β-D-maltopyranoside 5.4 (1.8) G11 UDαMn-Undecyl-α-D-maltopyranoside 5.8 (0.58) G12 UDMn-Undecyl-β-D-maltopyranoside 5.9 (0.59) H1 ωUDMω-Undecylenyl-β-D-maltopyranoside 3.6 (1.2) H2 DDαMn-Dodecyl-α-D-maltopyranoside 7.5 (0.15) H3 DDMn-Dodecyl-β-D-maltopyranoside 8.5 (0.17) H4 TDMn-Tridecyl-β-D-maltopyranoside 1.5 (0.03) H5 OTMn-Octyl-β-D-thiomaltopyranoside 21.25 (8.5) H6 NTMn-Nonyl-β-D-thiomaltopyranoside 9.6 (3.2) H7 DTMn-Decyl-β-D-thiomaltopyranoside 9 (0.9) H8 UDTMn-Undecyl-β-D-thiomaltopyranoside 10.5 (0.21) H9 DDTMn-Dodecyl-β-D-thiomaltopyranoside 5 (0.05) H10 S-8 Sucrose8 48.8 (24.4)H11 S-10 Sucrose10 7.5 (2.5) H12 S-12 Sucrose12 15 (0.3)


17. The method of claim 1, wherein said solution comprising said proteinin a first detergent comprises about 1000 micrograms or less of saidprotein.
 18. The method of claim 1, wherein said solution comprisingsaid protein in a first detergent comprises about 500 micrograms or lessof said protein.
 19. The method of claim 1, wherein said solutioncomprising said protein in a first detergent comprises about 400micrograms or less of said protein.
 20. The method of claim 1, whereinsaid solution comprising said protein in a first detergent comprisesabout 200 micrograms or less of said protein.
 21. The method of claim 1,wherein said solution comprising said protein in a first detergentcomprises about 100 micrograms or less of said protein.
 22. The methodof claim 1, wherein said solution comprising said protein in a firstdetergent comprises about 50 micrograms or less of said protein.
 23. Themethod of claim 1, wherein said method is performed in less than abouttwo hours.
 24. The method of claim 1, wherein said method is performedin less than about 1 hour.
 25. The method of claim 1, wherein saidprotein amounts are determined by dot blot or Western blot analysis. 26.The method of claim 25, wherein said protein amounts determined from thehigh molecular weight cut-off dot blots are normalized and plotted withthe ratio of low:high normalized intensities and the values grouped intoquartiles.
 27. The method of claim 25, wherein said protein amountsdetermined from the high molecular weight cut-off dot blots arenormalized and plotted graphically on the abscissa while the ratio oflow:high normalized intensities are plotted on the ordinate.
 28. Themethod of claim 1, further wherein said method is used to screendetergent mixtures, additives, ionic strength, and pH.
 29. The method ofclaim 1, wherein said protein is a membrane protein.
 30. The method ofclaim 1, wherein the concentration of said different detergent is thecritical micelle concentration of said different detergent.
 31. Themethod of claim 30, wherein the critical micelle concentration of saiddifferent detergent is selected from the following table: Well Abbrev.Name [Det] mM A1 No detergent (−control) A2 Empty well for currentdetergent (+control) A3 Z3-12 ZWITTERGENT ® 3-12 8.4 (2.8) A4 Z3-14ZWITTERGENT ® 3-14 10 (0.2) A5 DMG n-Decyl-N,N-dimethylglycine 38 (19)A6 DOMG n-Dodecyl-N,N-dimethylglycine 4.5 (1.5) A7 DAOn-Decyl-N,N-dimethylamine-N-oxide 21 (10.5) A8 UDAOn-Undecyl-N,N,-dimethylamine-N-oxide 9.6 (3.2) A9 LDAOn-Dodecyl-N,N-dimethylamine-N-oxide 3 (1) A10 C-DDFOS C-DODECAFOS ™ 44(22) A11 CF-4 CYCLOFOS ™-4 28 (14) A12 CF-5 CYCLOFOS ™-5 13.5 (4.5) B1CF-6 CYCLOFOS ™-6 8.04 (2.68) B2 CF-7 CYCLOFOS ™-7 6.2 (0.62) B3 FC-10FOS-CHOLINE ®-10 22 (11) B4 FC-11 FOS-CHOLINE ®-11 5.55 (1.85) B5 FC-12FOS-CHOLINE ®-12 4.5 (1.5) B6 FC-13 FOS-CHOLINE ®-13 7.5 (0.75) B7 FC-14FOS-CHOLINE ®-14 6 (0.12) B8 FC-I11 FOS-CHOLINE ®-ISO-11 53.2 (26.6) B9FC-I11-6U FOS-CHOLINE ®-ISO-11-6U 51.6 (25.8) B10 FC-I9FOS-CHOLINE ®-ISO-9 64 (32) B11 FC-U10-11 FOS-CHOLINE ®-UNSAT-11-10 15.5(6.2) B12 DHPC 1,2-Diheptanoyl-sn-glycero-3-phosphocholine 4.2 (1.4) C1LPC-10 LysoPC-10 20 (8) C2 LPC-12 LysoPC-12 7 (0.7) C3 FOSFEN-9FOSFEN ™-9 4.05 (1.35) C4 CHAPS CHAPS 20 (8) C5 CHAPSO CHAPSO 20 (8) C6DDMAU n-Dodecyl-N,N-(dimethylammonio)undecanoate 6.5 (0.13) C7 DDMABn-Dodecyl-N,N-(dimethylammonio)butyrate 12.9 (4.3) C8 LAPAO LAPAO 4.8(1.6) C9 TRIPAO TRIPAO 13.5 (4.5) C10 T-20 TWEEN ® 20 5.9 (0.059) C11BRIJ-35 BRIJ ® 35 9.1 (0.091) C12 TX-100 TRITON ® X-100 11.5 (0.23) D1TX-114 TRITON ® X-114 10 (0.2) D2 TX-305 TRITON ® X-305 6.5 (0.65) D3TX-405 TRITON ® X-405 8.1 (0.81) D4 NID-P40[Octylphenoxy]polyethoxyethanol 15 (0.3) D5 APO8 Dimethyloctylphosphineoxide 80 (40) D6 APO9 Dimethylnonylphosphine oxide 20 (10) D7 APO10Dimethyldecylphosphine oxide 13.98 (4.66) D8 APO11Dimethylundecylphosphine oxide 3.6 (1.2) D9 APO12Dimethyldodecylphosphine oxide 5.7 (0.57) D10 C6E3 Triethylene glycolmonohexyl ether 46 (23) D11 C6E4 Tetraethylene glycol monohexyl ether 60(30) D12 C6E5 Pentaethylene glycol monohexyl ether 74 (37) E1 C7E5Pentaethylene glycol monoheptyl ether 42 (21) E2 C8E4 Tetraethyleneglycol monooctyl ether 20 (8) E3 C8E5 Pentaethylene glycol monooctylether 17.75 (7.1) E4 C8E6 Hexaethylene glycol monooctyl ether 25 (10) E5C10E5 Pentaethylene glycol monodecyl ether 8.1 (0.81) E6 C10E6Hexaethylene glycol monodecyl ether 9 (0.9) E7 C10E9Polyoxyethylene(9)decyl ether 3.9 (1.3) E8 C12E8 Octaethylene glycolmonododecyl ether 9 (0.09) E9 C12E9 Polyoxyethylene(9)dodecyl ether 5(0.05) E10 C12E10 Polyoxyethylene(10)dodecyl ether 10 (0.2) E11 C13E8Polyoxyethylene(8)tridecyl ether 10 (0.1) E12 CHAP Big CHAP 8.7 (2.9) F1CHAP-D Big CHAP, deoxy 4.2 (1.4) F2 OHES Octyl-2-hydroxyethyl-sulfoxide48.4 (24.2) F3 RDHPOS Rac-2,3-dihydroxypropyloctylsulfoxide 48.4 (24.2)F4 GX-100 Genapol ® X-100 7.5 (0.15) F5 HTGn-Heptyl-β-D-thioglucopyranoside 58 (29) F6 OGn-Octyl-β-D-glucopyranoside 36 (18) F7 NG n-Nonyl-β-D-glucopyranoside16.25 (6.5) F8 CYGLU-3 CYGLU ®-3 56 (28) F9 HECAMEG HECAMEG 39 (19.5)F10 HEGA-9 Hega ®-9 78 (39) F11 C-HEGA-10 C-Hega ®-10 70 (35) F12C-HEGA-11 C-Hega ®-11 23 (11.5) G1 CYMAL-3 CYMAL ®-3 60 (30) G2 CYMAL-4CYMAL ®-4 19 (7.6) G3 CYMAL-5 CYMAL ®-5 7.2 (2.4) G4 CYMAL-6 CYMAL ®-65.6 (0.56) G5 CYMAL-7 CYMAL ®-7 9.5 (0.19) G6 DMHM2,6-Dimethyl-4-heptyl-β-D-maltoside 55 (27.5) G7 OMn-Octyl-β-D-maltopyranoside 39 (19.5) G8 NM n-Nonyl-β-D-maltopyranoside15 (6) G9 DαM n-Decyl-α-D-maltopyranoside 4.8 (1.6) G10 DMn-Decyl-β-D-maltopyranoside 5.4 (1.8) G11 UDαMn-Undecyl-α-D-maltopyranoside 5.8 (0.58) G12 UDMn-Undecyl-β-D-maltopyranoside 5.9 (0.59) H1 ωUDMω-Undecylenyl-β-D-maltopyranoside 3.6 (1.2) H2 DDαMn-Dodecyl-α-D-maltopyranoside 7.5 (0.15) H3 DDMn-Dodecyl-β-D-maltopyranoside 8.5 (0.17) H4 TDMn-Tridecyl-β-D-maltopyranoside 1.5 (0.03) H5 OTMn-Octyl-β-D-thiomaltopyranoside 21.25 (8.5) H6 NTMn-Nonyl-β-D-thiomaltopyranoside 9.6 (3.2) H7 DTMn-Decyl-β-D-thiomaltopyranoside 9 (0.9) H8 UDTMn-Undecyl-β-D-thiomaltopyranoside 10.5 (0.21) H9 DDTMn-Dodecyl-β-D-thiomaltopyranoside 5 (0.05) H10 S-8 Sucrose8 48.8 (24.4)H11 S-10 Sucrose10 7.5 (2.5) H12 S-12 Sucrose12 15 (0.3)


32. The method of claim 1, wherein said different detergent has moderateor high aqueous solubility.
 33. The method of claim 1, wherein saiddifferent detergent has zwitterionic or nonionic headgroups.
 34. Adetergent panel for determining the stability and size of a protein,said panel comprising at least two detergents at or above their criticalmicelle concentrations, and optionally a positive control and a negativecontrol, wherein said detergents and their critical micelleconcentrations, or suitably higher concentrations, are selected from thefollowing table: Well Abbrev. Name [Det] mM A1 No detergent (−control)A2 Empty well for current detergent (+control) A3 Z3-12 ZWITTERGENT ®3-12 8.4 (2.8) A4 Z3-14 ZWITTERGENT ® 3-14 10 (0.2) A5 DMGn-Decyl-N,N-dimethylglycine 38 (19) A6 DOMGn-Dodecyl-N,N-dimethylglycine 4.5 (1.5) A7 DAOn-Decyl-N,N-dimethylamine-N-oxide 21 (10.5) A8 UDAOn-Undecyl-N,N,-dimethylamine-N-oxide 9.6 (3.2) A9 LDAOn-Dodecyl-N,N-dimethylamine-N-oxide 3 (1) A10 C-DDFOS C-DODECAFOS ™ 44(22) A11 CF-4 CYCLOFOS ™-4 28 (14) A12 CF-5 CYCLOFOS ™-5 13.5 (4.5) B1CF-6 CYCLOFOS ™-6 8.04 (2.68) B2 CF-7 CYCLOFOS ™-7 6.2 (0.62) B3 FC-10FOS-CHOLINE ®-10 22 (11) B4 FC-11 FOS-CHOLINE ®-11 5.55 (1.85) B5 FC-12FOS-CHOLINE ®-12 4.5 (1.5) B6 FC-13 FOS-CHOLINE ®-13 7.5 (0.75) B7 FC-14FOS-CHOLINE ®-14 6 (0.12) B8 FC-I11 FOS-CHOLINE ®-ISO-11 53.2 (26.6) B9FC-I11-6U FOS-CHOLINE ®-ISO-11-6U 51.6 (25.8) B10 FC-I9FOS-CHOLINE ®-ISO-9 64 (32) B11 FC-U10-11 FOS-CHOLINE ®-UNSAT-11-10 15.5(6.2) B12 DHPC 1,2-Diheptanoyl-sn-glycero-3-phosphocholine 4.2 (1.4) C1LPC-10 LysoPC-10 20 (8) C2 LPC-12 LysoPC-12 7 (0.7) C3 FOSFEN-9FOSFEN ™-9 4.05 (1.35) C4 CHAPS CHAPS 20 (8) C5 CHAPSO CHAPSO 20 (8) C6DDMAU n-Dodecyl-N,N-(dimethylammonio)undecanoate 6.5 (0.13) C7 DDMABn-Dodecyl-N,N-(dimethylammonio)butyrate 12.9 (4.3) C8 LAPAO LAPAO 4.8(1.6) C9 TRIPAO TRIPAO 13.5 (4.5) C10 T-20 TWEEN ® 20 5.9 (0.059) C11BRIJ-35 BRIJ ®35 9.1 (0.091) C12 TX-100 TRITON ® X-100 11.5 (0.23) D1TX-114 TRITON ® X-114 10 (0.2) D2 TX-305 TRITON ® X-305 6.5 (0.65) D3TX-405 TRITON ® X-405 8.1 (0.81) D4 NID-P40[Octylphenoxy]polyethoxyethanol 15 (0.3) D5 APO8 Dimethyloctylphosphineoxide 80 (40) D6 APO9 Dimethylnonylphosphine oxide 20 (10) D7 APO10Dimethyldecylphosphine oxide 13.98 (4.66) D8 APO11Dimethylundecylphosphine oxide 3.6 (1.2) D9 APO12Dimethyldodecylphosphine oxide 5.7 (0.57) D10 C6E3 Triethylene glycolmonohexyl ether 46 (23) D11 C6E4 Tetraethylene glycol monohexyl ether 60(30) D12 C6E5 Pentaethylene glycol monohexyl ether 74 (37) E1 C7E5Pentaethylene glycol monoheptyl ether 42 (21) E2 C8E4 Tetraethyleneglycol monooctyl ether 20 (8) E3 C8E5 Pentaethylene glycol monooctylether 17.75 (7.1) E4 C8E6 Hexaethylene glycol monooctyl ether 25 (10) E5C10E5 Pentaethylene glycol monodecyl ether 8.1 (0.81) E6 C10E6Hexaethylene glycol monodecyl ether 9 (0.9) E7 C10E9Polyoxyethylene(9)decyl ether 3.9 (1.3) E8 C12E8 Octaethylene glycolmonododecyl ether 9 (0.09) E9 C12E9 Polyoxyethylene(9)dodecyl ether 5(0.05) E10 C12E10 Polyoxyethylene(10)dodecyl ether 10 (0.2) E11 C13E8Polyoxyethylene(8)tridecyl ether 10 (0.1) E12 CHAP Big CHAP 8.7 (2.9) F1CHAP-D Big CHAP, deoxy 4.2 (1.4) F2 OHES Octyl-2-hydroxyethyl-sulfoxide48.4 (24.2) F3 RDHPOS Rac-2,3-dihydroxypropyloctylsulfoxide 48.4 (24.2)F4 GX-100 Genapol ® X-100 7.5 (0.15) F5 HTGn-Heptyl-β-D-thioglucopyranoside 58 (29) F6 OGn-Octyl-β-D-glucopyranoside 36 (18) F7 NG n-Nonyl-β-D-glucopyranoside16.25 (6.5) F8 CYGLU-3 CYGLU ®-3 56 (28) F9 HECAMEG HECAMEG 39 (19.5)F10 HEGA-9 Hega ®-9 78 (39) F11 C-HEGA-10 C-Hega ®-10 70 (35) F12C-HEGA-11 C-Hega ®-11 23 (11.5) G1 CYMAL-3 CYMAL ®-3 60 (30) G2 CYMAL-4CYMAL ®-4 19 (7.6) G3 CYMAL-5 CYMAL ®-5 7.2 (2.4) G4 CYMAL-6 CYMAL ®-65.6 (0.56) G5 CYMAL-7 CYMAL ®-7 9.5 (0.19) G6 DMHM2,6-Dimethyl-4-heptyl-β-D-maltoside 55 (27.5) G7 OMn-Octyl-β-D-maltopyranoside 39 (19.5) G8 NM n-Nonyl-β-D-maltopyranoside15 (6) G9 DαM n-Decyl-α-D-maltopyranoside 4.8 (1.6) G10 DMn-Decyl-β-D-maltopyranoside 5.4 (1.8) G11 UDαMn-Undecyl-α-D-maltopyranoside 5.8 (0.58) G12 UDMn-Undecyl-β-D-maltopyranoside 5.9 (0.59) H1 ωUDMω-Undecylenyl-β-D-maltopyranoside 3.6 (1.2) H2 DDαMn-Dodecyl-α-D-maltopyranoside 7.5 (0.15) H3 DDMn-Dodecyl-β-D-maltopyranoside 8.5 (0.17) H4 TDMn-Tridecyl-β-D-maltopyranoside 1.5 (0.03) H5 OTMn-Octyl-β-D-thiomaltopyranoside 21.25 (8.5) H6 NTMn-Nonyl-β-D-thiomaltopyranoside 9.6 (3.2) H7 DTMn-Decyl-β-D-thiomaltopyranoside 9 (0.9) H8 UDTMn-Undecyl-β-D-thiomaltopyranoside 10.5 (0.21) H9 DDTMn-Dodecyl-β-D-thiomaltopyranoside 5 (0.05) H10 S-8 Sucrose8 48.8 (24.4)H11 S-10 Sucrose10 7.5 (2.5) H12 S-12 Sucrose12 15 (0.3)


35. The detergent panel of claim 34, wherein all 96 detergents andcontrols are used.
 36. A kit for determining the stability and size of aprotein, said kit comprising at least one detergent, at least onechamber wherein said chamber comprises a filter of about 0.2 μm poresize, at least one chamber comprising a high molecular weight cut-offfilter, at least one chamber comprising a low molecular weight cut-offfilter, optionally at least one standard protein, and an instructionalmaterial for the used thereof.
 37. The kit of claim 36, wherein saidchambers are wells in 96 well plates, said high molecular weight cut-offfilter is about 300 kDa, and said low molecular weight cut-off filter isabout 100 kDa.
 38. The kit of claim 36, further comprising at least onedetergent or control selected from the following table: Well Abbrev.Name [Det] mM A1 No detergent (−control) A2 Empty well for currentdetergent (+control) A3 Z3-12 ZWITTERGENT ® 3-12 8.4 (2.8) A4 Z3-14ZWITTERGENT ® 3-14 10 (0.2) A5 DMG n-Decyl-N,N-dimethylglycine 38 (19)A6 DOMG n-Dodecyl-N,N-dimethylglycine 4.5 (1.5) A7 DAOn-Decyl-N,N-dimethylamine-N-oxide 21 (10.5) A8 UDAOn-Undecyl-N,N,-dimethylamine-N-oxide 9.6 (3.2) A9 LDAOn-Dodecyl-N,N-dimethylamine-N-oxide 3 (1) A10 C-DDFOS C-DODECAFOS ™ 44(22) A11 CF-4 CYCLOFOS ™-4 28 (14) A12 CF-5 CYCLOFOS ™-5 13.5 (4.5) B1CF-6 CYCLOFOS ™-6 8.04 (2.68) B2 CF-7 CYCLOFOS ™-7 6.2 (0.62) B3 FC-10FOS-CHOLINE ®-10 22 (11) B4 FC-11 FOS-CHOLINE ®-11 5.55 (1.85) B5 FC-12FOS-CHOLINE ®-12 4.5 (1.5) B6 FC-13 FOS-CHOLINE ®-13 7.5 (0.75) B7 FC-14FOS-CHOLINE ®-14 6 (0.12) B8 FC-I11 FOS-CHOLINE ®-ISO-11 53.2 (26.6) B9FC-I11-6U FOS-CHOLINE ®-ISO-11-6U 51.6 (25.8) B10 FC-I9FOS-CHOLINE ®-ISO-9 64 (32) B11 FC-U10-11 FOS-CHOLINE ®-UNSAT-11-10 15.5(6.2) B12 DHPC 1,2-Diheptanoyl-sn-glycero-3-phosphocholine 4.2 (1.4) C1LPC-10 LysoPC-10 20 (8) C2 LPC-12 LysoPC-12 7 (0.7) C3 FOSFEN-9FOSFEN ™-9 4.05 (1.35) C4 CHAPS CHAPS 20 (8) C5 CHAPSO CHAPSO 20 (8) C6DDMAU n-Dodecyl-N,N-(dimethylammonio)undecanoate 6.5 (0.13) C7 DDMABn-Dodecyl-N,N-(dimethylammonio)butyrate 12.9 (4.3) C8 LAPAO LAPAO 4.8(1.6) C9 TRIPAO TRIPAO 13.5 (4.5) C10 T-20 TWEEN ® 20 5.9 (0.059) C11BRIJ-35 BRIJ ®35 9.1 (0.091) C12 TX-100 TRITON ® X-100 11.5 (0.23) D1TX-114 TRITON ® X-114 10 (0.2) D2 TX-305 TRITON ® X-305 6.5 (0.65) D3TX-405 TRITON ® X-405 8.1 (0.81) D4 NID-P40[Octylphenoxy]polyethoxyethanol 15 (0.3) D5 APO8 Dimethyloctylphosphineoxide 80 (40) D6 APO9 Dimethylnonylphosphine oxide 20 (10) D7 APO10Dimethyldecylphosphine oxide 13.98 (4.66) D8 APO11Dimethylundecylphosphine oxide 3.6 (1.2) D9 APO12Dimethyldodecylphosphine oxide 5.7 (0.57) D10 C6E3 Triethylene glycolmonohexyl ether 46 (23) D11 C6E4 Tetraethylene glycol monohexyl ether 60(30) D12 C6E5 Pentaethylene glycol monohexyl ether 74 (37) E1 C7E5Pentaethylene glycol monoheptyl ether 42 (21) E2 C8E4 Tetraethyleneglycol monooctyl ether 20 (8) E3 C8E5 Pentaethylene glycol monooctylether 17.75 (7.1) E4 C8E6 Hexaethylene glycol monooctyl ether 25 (10) E5C10E5 Pentaethylene glycol monodecyl ether 8.1 (0.81) E6 C10E6Hexaethylene glycol monodecyl ether 9 (0.9) E7 C10E9Polyoxyethylene(9)decyl ether 3.9 (1.3) E8 C12E8 Octaethylene glycolmonododecyl ether 9 (0.09) E9 C12E9 Polyoxyethylene(9)dodecyl ether 5(0.05) E10 C12E10 Polyoxyethylene(10)dodecyl ether 10 (0.2) E11 C13E8Polyoxyethylene(8)tridecyl ether 10 (0.1) E12 CHAP Big CHAP 8.7 (2.9) F1CHAP-D Big CHAP, deoxy 4.2 (1.4) F2 OHES Octyl-2-hydroxyethyl-sulfoxide48.4 (24.2) F3 RDHPOS Rac-2,3-dihydroxypropyloctylsulfoxide 48.4 (24.2)F4 GX-100 Genapol ® X-100 7.5 (0.15) F5 HTGn-Heptyl-β-D-thioglucopyranoside 58 (29) F6 OGn-Octyl-β-D-glucopyranoside 36 (18) F7 NG n-Nonyl-β-D-glucopyranoside16.25 (6.5) F8 CYGLU-3 CYGLU ®-3 56 (28) F9 HECAMEG HECAMEG 39 (19.5)F10 HEGA-9 Hega ®-9 78 (39) F11 C-HEGA-10 C-Hega ®-10 70 (35) F12C-HEGA-11 C-Hega ®-11 23 (11.5) G1 CYMAL-3 CYMAL ®-3 60 (30) G2 CYMAL-4CYMAL ®-4 19 (7.6) G3 CYMAL-5 CYMAL ®-5 7.2 (2.4) G4 CYMAL-6 CYMAL ®-65.6 (0.56) G5 CYMAL-7 CYMAL ®-7 9.5 (0.19) G6 DMHM2,6-Dimethyl-4-heptyl-β-D-maltoside 55 (27.5) G7 OMn-Octyl-β-D-maltopyranoside 39 (19.5) G8 NM n-Nonyl-β-D-maltopyranoside15 (6) G9 DαM n-Decyl-α-D-maltopyranoside 4.8 (1.6) G10 DMn-Decyl-β-D-maltopyranoside 5.4 (1.8) G11 UDαMn-Undecyl-α-D-maltopyranoside 5.8 (0.58) G12 UDMn-Undecyl-β-D-maltopyranoside 5.9 (0.59) H1 ωUDMω-Undecylenyl-β-D-maltopyranoside 3.6 (1.2) H2 DDαMn-Dodecyl-α-D-maltopyranoside 7.5 (0.15) H3 DDMn-Dodecyl-β-D-maltopyranoside 8.5 (0.17) H4 TDMn-Tridecyl-β-D-maltopyranoside 1.5 (0.03) H5 OTMn-Octyl-β-D-thiomaltopyranoside 21.25 (8.5) H6 NTMn-Nonyl-β-D-thiomaltopyranoside 9.6 (3.2) H7 DTMn-Decyl-β-D-thiomaltopyranoside 9 (0.9) H8 UDTMn-Undecyl-β-D-thiomaltopyranoside 10.5 (0.21) H9 DDTMn-Dodecyl-β-D-thiomaltopyranoside 5 (0.05) H10 S-8 Sucrose8 48.8 (24.4)H11 S-10 Sucrose10 7.5 (2.5) H12 S-12 Sucrose12 15 (0.3)


39. The kit of claim 38, wherein said kit comprises at least one test 96well plate comprising all of the detergents and controls according tosaid table.
 40. The method of claim 1, wherein at least one of saidfirst, second, or third chambers is subjected to centrifugation toenhance the filtration process.